Pharmaceutical compositions with resistance to soluble CEA

ABSTRACT

The present disclosure relates to a bispecific single chain antibody which has a first binding domain specifically binding to human CD3, and a second binding domain specifically binding to human CEA, where the second binding domain comprises at least a part of the CDR-H3 or the complete CDR-H3 of murine monoclonal antibody A5B7, a pharmaceutical composition comprising the bispecific single chain antibody, and methods for the treatment of an epithelial tumor in a human with the pharmaceutical compositions containing the bispecific single chain antibody. Furthermore, processes for the production of the pharmaceutical compositions as well as medical/pharmaceutical uses for the specific bispecific single chain antibody molecules bearing specificities for the human CD3 antigen and the human CEA antigen are disclosed.

More than three decades have passed since Gold and Freedman firstdescribed the tumor associated carcinoembryonic antigen (CEA) in humancolon cancer tissue extracts (Gold and Freedman; J. Exp. Med. 122(1965); 467-481).

Meanwhile, 28 other genes/pseudogenes relating to the CEA gene familyhave been discovered. In an attempt to simplify the nomenclature usedfor the members of the CEA gene family, the family has recently beenrenamed the “CEA-related cellular adhesion molecules” (CEACAMs) and thenomenclature for its members has been unified (Beauchemin, Exp. CellRes. 252 (1999), 243-249). For example, according to this nomenclature,human CEA (CD66e) is termed CEACAM5.

The human CEA gene family is clustered on chromosome 19q13.2 (Olsen etal. Genomics 23 (1994); 659-668). Its 29 genes and pseudogenes can bedivided into three subgroups, i.e. the CEA subgroup containing sevenexpressed genes, the pregnancy-specific-glycoprotein (PSG) subgroupcontaining eleven expressed genes and the third subgroup which containsonly pseudogenes (Hammarström, Sem. Cancer Biol. 9 (1999), 67-81;Beauchemin, Exp. Cell Res. 252 (1999), 243-249). The analysis of theamino acid sequences of CEA and the other family members revealed thatthey belong to the immunoglobulin (Ig) superfamily (Williams andBarclay, Annul. Rev. Immunol. 6 (1988), 381-405). All members of the CEAsubgroup are attached to the cell surface membrane: Biliary glycoprotein(CEACAM1; BGP1; TM-CEA; CD66a), CEA gene family member 1 (CEACAM3; CGM1;CD66d) and CEA gene family member 7 (CEACAM4; CGM7) have hydrophobictransmembrane domains, whereas carcinoembryonic antigen(carcinoembryonic antigen-related cell adhesion molecule 5; CEACAM5;CEA; CD66e), non-specific cross-reacting antigen (CEACAM6; NCA;NCA-50/90; CD66c), CEA gene family member 2 (CEACAM7; CGM2) and CEA genefamily member 6 (CEACAM8; CGM6; CD66b) are linked to the plasma membraneby glycosylphosphatidylinositol (GPI) lipid moieties. The CEA proteinsare highly glycosylated with a molecular weight of up to approximately300 kDa, depending on the number of Ig domains.

As regards the biological activity of the CEA proteins, in vitro studieswith tumor cell lines suggested that several CEA subfamilies includingbiliary glycoprotein, CEA and non-specific cross-reacting antigen canact as homophilic and heterotypic cell adhesion molecules when expressedon the tumor cell surface (Oikawa et al., Biochem. Biophys. Res. Commun.186 (1992), 881-887; Zhou et al., Cancer Res. 53 (1993), 3817-3822).More recently, a possible role of CEA and non-specific cross-reactingantigen in the innate immune defense protecting colon from microbialattack has been discussed (Hammarström and Baranov, Trends Microbiol. 9(2001), p. 119-125). In particular, it has been proposed that theseproteins bind and trap microorganisms preventing them from reaching andinvading the epithelial cells of the microvilli.

It was hypothesized that CEA is an oncofetal antigen which is expressedduring fetal life, absent in the healthy adult and re-expressed incancer. However, CEA is also expressed in normal adult tissue. Forinstance, biliary glycoprotein, CEA, non-specific cross-reacting antigenand CEA gene family member 2 are expressed in normal human colon,particularly in the mature columnar epithelial cells facing the gutlumen and in the highly differentiated cells at the crypt mouth(Frängsmyr et al., Cancer Res. 55 (1995), 2963-2967; Frängsmyr et al.,Tumor Biol. 20 (1999), 277-292). More specifically, these proteins arelocalized to the brush-border glycocalyx of the mature colonocyteslining the free luminal surface. Biliary glycoprotein, CEA andnon-specific cross-reacting antigen are also expressed in a number oftumors of epithelial origin (Hammarström, Sem. Cancer Biol. 9 (1999),67-81; Shively and Beatty CRC Crit. Rev. Oncol. Hematol. 2 (1985),355-399).

Already in the late 1970s and early 1980s, CEA became a favored targetantigen for radioimmunolocalization of colorectal and other epithelialtumors. This is due to the fact that CEA is overexpressed in 95% ofgastrointestinal and pancreatic cancers, as well as in most small-celland non-small-cell lung carcinomas. It is also expressed in breastcarcinoma and squamous cell carcinoma of the head and neck (Primus etal., Cancer 42 (1978), 1540-1545). In fact, CEA is one of the mostextensively used clinical tumor markers. It is used as a serum tumormarker for colorectal and some other cancers due to its stability, itsfairly restricted expression in normal adult tissue and its highexpression in tumors of epithelial origin. The bulk of CEA in a healthyindividual is produced in colon. There it is released from the apicalsurface of mature columnar cells into the gut lumen and disappears withthe feces. Thus, only very low levels are normally seen in the bloodfrom healthy individuals. For instance, CEA levels in the blood ofhealthy individuals is less than 2 μg/l. In contrast, CEA levels inserum from patients with colorectal and other carcinomas are increased,ranging up to more than 2000 μg/l (Thomson et al., PNAS 64 (1969),161-167). In particular, progressive, malignant, or late stageepithelial tumors are frequently accompanied by high serumconcentrations of soluble CEA (Fletcher; Ann. Intern. Med. 104 (1986),66-73). It is known that components from the plasma membrane, includingCEA, are continually exfoliated from the surface as plasmamembrane-derived vesicles (Taylor and Black, J. Natl. Cancer Inst. 74(1985), 859-866; Sack et al., J Clin Invest. 82 (1988), 586-93) whichthrough draining lymph and blood vessels can end up in the blood. As thetumor size increases, more CEA will accumulate in the blood. The mainuse of serum CEA determinations as a tumor marker is in thepost-surgical surveillance of colon cancer. Increased CEA levels was thefirst indicator of recurrent disease in 81% (Minton et al., Cancer 55(1985), 1284-1290) and 89% (Wanebo et al., Surg. Gynecol. Obstet. 169(1989), 479-487) of patients, respectively. Serum CEA levels can also beused as a prognostic indicator (Mulcahy and Benson, Curr. Oncol. Rep. 1(1999), 168-172).

Due to its over-expression in many epithelial cancers CEA is not onlyused as a tumor marker but also as a target for anti-tumor therapy. Forexample, gastrointestinal cancers account for a large proportion ofhuman epithelial tumors, with an estimated 21.700 new cases of gastriccancer and 135.400 new cases of colorectal cancer in the United Statesin the year 2001 (Greenlee; CA Cancer J Clin 51 (2001), 15-36).Colorectal cancer is the third most common malignancy and the thirdleading cause of death from cancer in both males and females (Ries;Cancer 88 (2000), 2398-2424). In an attempt to find new therapeuticsagainst these tumors, anti-CEA monoclonal antibodies have been exploredas possible therapeutics for CEA-positive cancers (Murakami et al.,Immunol. Invest. 25 (1996), 23-35).

One example for an approach in which patients with low tumor load(corresponding to low serum CEA levels) have been successfully treatedis a study performed by Behr et al. In this approach, a ¹³¹I-labeledvariant of labetuzumab (labetuzumab is a humanized form of anti-CEAmonoclonal antibody MN-14; Behr et al., Cancer, 94: 1373-1381, (2002),1559-64) has been analysed in a phase II trial in which 30 CRC patientswith small volume metastatic disease chemorefractory to 5-fluorouraciland folinic acid or in an adjuvant setting after liver metastasis havebeen enrolled. A single injection of ¹³¹I-labeled labetuzumab was given.Of 19 assessable patients, 3 had partial remissions and eight showedminor responses up to 15 months in duration. In the adjuvant setting, 7of 9 patients were disease free for up to 3 years, whereas the relapserate in the control group was 67% in the same time period. The serum CEAlevels of the patients ranged from 3.9-45 ng/ml (Behr et al., Cancer,94: 1373-1381, 2002). In another study characterized by patients withlow CEA serum levels (<5 ng/ml), CEA radio-immunotherapy with ¹³¹Ilabetuzumab (loc. cit.) has been shown to improve survival post salvageresection of colorectal cancer metastases in the liver. 23 patientsreceived a dose of 40-60 mCi/m² ¹³¹I-labetuzumab. Five-year survival was51.3% for treated and 7.4% for control groups, respectively (Liersch etal., JCO, 2005, ASCO Proc, Vol 23, No 16S: 3627).

Yet, therapeutic approaches dealing with high serum CEA concentrationsfrequently resulted in low or no anti-tumor responses. For example, in aclinical study performed to evaluate a humanized anti-CEA monoclonalantibody in clinic, a CDR-grafted version of MN-14 (hMN-14; Sharkey,Cancer Res. 55 (23 Suppl) (1995) 5935s-5945s.) has been labeled with¹³¹I. 19 patients with advanced CEA-producing tumors received ¹³¹Ilabeled hMN-14. The biodistribution, tumor targeting, andpharmacokinetic behavior of the hMN-14 was similar to that seen with themurine MN-14. However, patients with elevated CEA (>200 ng/ml) in plasmahad more than 30% of the labeled antibody complexed within 1 h afterinjection. In some of these patients, increased complication resulted inenhanced metabolism of the antibody with more rapid clearance from theblood than that seen in patients with lower plasma CEA (Sharkey, loc.cit.). In another phase I trial carried out by Yu et al., an¹³¹I-labeled high-affinity murine monoclonal antibody (mAb) against CEA,COL-1 (Muraro, Cancer Res. 45 (1985), 5769-80), has been investigated inpatients with gastrointestinal malignancies. In particular, theinfluence of serum CEA and tumor bulk on pharmacokinetics has beenanalysed. To this end, 18 patients with advanced gastrointestinalmalignancies received 20 mg of COL-1 labeled with ¹³¹I, with doses from10 mCi/m² to 75 mCi/m². Serum CEA level ranged from 6 to 2739 ng/mL(mean+/−SD, 500+/−639). 82% of all tumor-involved organs were positiveand 58% of all lesions. However, it has been again observed thatelevated serum CEA (>500 ng/mL) and tumor bulk directly correlated withclearance of serum radioactivity. The authors concluded that patientswith highly elevated circulating CEA levels and/or increased tumor bulkclear ¹³¹I-labeled COL-1 more rapidly from the circulation (Yu et al.,J. Cli. Oncol. 14 (1996), 1798-1809). Similar results have been obtainedin a study by Hajjar et al. with iodine-131-labeled humanized MN-14anti-CEA monoclonal antibody in patients with metastaticgastrointestinal and colorectal cancer. In this phase I trial, 21patients either after prior external beam radiation or after standardchemotherapy have been treated with antibody. 7 of 21 patients had humananti-human antibodies (HAHAs), but no adverse effects. No antitumorresponse was observed. Again it has been found that elevated plasma CEAlevels increase the clearance of the antibody from the blood and wholebody (Hajjar et al., Clin Colorectal Cancer, 2 (2002), 31-42) which atleast in part may provide for an explanation of the lacking anti-tumorresponse observed in this study. The phenomenon of rapid clearance oftherapeutic antibody from blood and body may be explained by increasedformation of immune complexes which have to be rapidly removed from thebody in order to prevent organ damage. No therapeutic effect could beobserved in the tumor patients enrolled in these studies, most probablydue to the rapid clearance of the monoclonal antibodies.

In order to circumvent problems caused by immune complex formation andrapid clearance of therapeutic monoclonal antibodies which are mostlikely mediated by the Fc part of the antibodies recognized by Fcreceptors of immune cells, antibody derivatives (e.g. scFv constructs)or fragments (e.g. Fab and Fab₂ fragments) without Fc part have beenproduced and analysed in clinic. Most of these studies are directed totumor imaging and detection/localization (see e.g. Chester et al.,Cancer Chemother Pharmacol, 46 (2000) Suppl: S8-12; Mayer et al., ClinCancer Res, 6: (2000) 1711-1719; Begent et al., Nat Med, 2 (1996):979-984). Only a few studies investigated the therapeutic efficacy ofsuch antibody derivatives/fragments in clinic. For example, in aclinical approach by Francis et al. (Francis, Br. J. Cancer 87(6)(2002), 600-607) anti-tumor activity of a scFv-carboxypeptidaseconstruct has been investigated. In this phase I trial, the antibodydirected enzyme prodrug therapy (ADEPT) has been used in patients withadvanced colorectal carcinoma or other CEA producing tumours. To thisend, A5CP, consisting of a F(ab)₂ fragment of a mouse monoclonalantibody to CEA (A5B7) linked to the bacterial enzyme carboxypeptidase(CPG2) as the antibody-enzyme targeting agent and ZD2767P, a bis-iodophenol mustard prodrug have been utilized. As a result, no clinical orradiological responses have been seen in the study. Pre-treatment serumCEA levels ranged up to 1000 ng/ml. These high CEA concentrations inserum of the treated tumor patients may be at least in part responsiblefor the lacking anti-tumor response observed in this study.

In view of the problems set forth above, the provision of means andmethods for efficient therapeutics for progressive, malignant, or latestage epithelial tumors is highly desirable.

Accordingly, one aspect of the invention relates to a pharmaceuticalcomposition, said pharmaceutical composition comprising a bispecificsingle chain antibody which has

(a) a first binding domain specifically binding to human CD3, and

(b) a second binding domain specifically binding to human CEA,

wherein said second binding domain comprises at least the amino acidsequence “DX₁X₂X₃X₄FYFDY” (SEQ ID NO. 65), wherein “X₁”, “X₂”, “X₃” or“X₄” represents any amino acid residue, and the amino acid residue “D”corresponds to Kabat position 95 of CDR-H3 of murine monoclonal antibodyA5B7 and the amino acid residues “FYFDY” correspond to Kabat positions100, 100a, 100b, 101, and 102, respectively, of CDR-H3 of murinemonoclonal antibody A5B7. In one embodiment, “X₁” represents “R”(Arginine), “F” (Phenylalanine), “M” (Methionine), “E” (Glutamic acid),or “T” (Threonine); “X₂” represents “G” (Glycine), “Y” (Tyrosine), “A”(Alanine), “D” (Aspartic acid), or “S” (Serine); “X₃” represents “L”(Leucine), “F” (Phenylalanine), “M” (Methionine), “E” (Glutamic acid),or “T” (Threonine); and “X₄” represents “R” (Arginine), “Y” (Tyrosine),“A” (Alanine), “D” (Aspartic acid), or “S” (Serine).

In an embodiment of the pharmaceutical composition of the invention,said second binding domain specific for human CEA of the bispecificsingle chain antibody defined herein comprises at least the amino acidsequence “DRGLRFYFDY” (SEQ ID NO. 66) corresponding to Kabat positions95-102 of the CDR-H3 of murine monoclonal antibody A5B7.

The present invention provides means and methods particularly suited forthe treatment of epithelial tumor patients with high soluble CEAconcentrations in their plasma. Such high soluble CEA concentrations arefound in the serum/plasma of epithelial tumor patients with progressivetumors, recurrent, metastatic, late stage tumors and for patients withhigh tumor load/burden. It has been found that bispecific single chainantibodies with a CEA binding domain comprising the amino acid sequence“DRGLRFYFDY” (SEQ ID NO. 66) not only bind to CEA-positive target cells,but also to soluble CEA; see Example 3 and FIG. 2 of the presentinvention; and EP B1 491031. The indicated amino acid sequence“DRGLRFYFDY” corresponds to Kabat positions 95-102 (SEQ ID NO. 66) ofthe CDR-H3 of murine monoclonal antibody A5B7 (Harwood, Br J Cancer. 54(1986), 75-82). Surprisingly, although binding to soluble CEA, saidbispecific single chain antibodies kill CEA-bearing tumor cells, even inthe presence of high concentrations of soluble CEA (up to 1 μg/mlsoluble CEA has been tested). Put in other words, said bispecificconstructs are not inhibited by soluble CEA in their cytotoxic activityagainst CEA-positive tumor cells.

As shown in the following Examples 5 and 8 (in combination with FIGS. 5,6, 8, 10, 19, 20, 22 and 27), bispecific single chain antibodies with aCEA binding domain comprising the amino acid sequence “DRGLRFYFDY”mediated cytotoxicity to CEA-positive tumor cells, in the presence ofeven high concentrations of soluble CEA. For instance, FIG. 10 shows acytotoxicity assay of CEA-reactive bispecific single chain constructsredirected to Kato III cells (CEA-positive human gastric carcinoma cellline) in the presence of increasing amounts of soluble CEA antigen.Stimulated human CD8 positive cytotoxic T cells (CTLs) were used aseffector cells. CEAI VHVL×SEQ ID NO.77 VHVL-mediated cytotoxicity isresistant to soluble CEA. In contrast, CEAII VHVL×SEQ ID NO.77VHVL-mediated cytotoxic activity is inhibited by increasing amounts ofsoluble CEA. CEAI is a variable region derived from murine mAb A5B7,whereas CEAII VHVL is derived from mAb T84.66.

Importantly, it has been found that the amino acid sequence “DRGLRFYFDY”is sufficient to mediate resistance to soluble CEA when used in a humanCEA-binding domain (i.e. a human binding domain specifically binding tohuman CEA) of anti-CEAxanti-CD3 bispecific single chain antibodies; seee.g. FIGS. 19, 20, 22 and 27.

In the following, bispecific single chain antibodies as defined hereinare therefore referred to as being resistant to soluble CEA antigen. Theterm “resistance to soluble CEA antigen”, “resistant to soluble CEA” orrelated terms as used herein refers to the fact that the cytotoxicityagainst CEA-positive target or tumor cells mediated by said bispecificsingle chain antibodies is not affected by increasing concentrations ofsoluble CEA. In particular, the cytotoxic activity is not inhibited byeven high concentrations of soluble CEA (up to 1 μg/ml has been tested).As set forth above, CEA levels in the blood of healthy individuals isless than 2 ng/ml. High soluble CEA concentrations in the serum/plasmaof tumor patients are characteristic for progressive, recurrent,metastatic, or late stage tumors and for patients with high tumor load.Thus, the present invention provides means and methods particularlysuited for the treatment of epithelial tumor patients with such highsoluble CEA concentrations in their plasma. The term “high soluble CEAconcentrations” as used herein denotes a soluble serum/plasma-CEAconcentration higher than 10, 20, 50, 70, 80, 90 or 100 ng/ml. Thisserum/plasma-CEA concentration may be determined, inter alia, by ELISA.Preferably, said soluble serum/plasma-CEA concentration is higher than100 ng/ml, as determined e.g. by ELISA.

The generation of said bispecific single chain antibodies withresistance to soluble CEA antigen was no trivial task, as evident fromthe following Examples. For instance, bispecific single chain antibodieswith a CEA binding domain derived from a monoclonal antibody (mAb) knownto bind membrane-bound CEA but not soluble CEA, i.e. mAb PR1A3 (Durbin,Proc Natl Acad Sci USA. 91 (1994), 4313-7), could not be produced: Whenused in the bispecific single chain antibody format, noexpression/secretion of the anti-CD3×anti-CEA bispecific single chainconstruct could be achieved. When a humanized version of PR1A3 (Durbin,loc. cit.) has been utilized for the generation, the bispecific singlechain antibody construct was expressed and secreted from the host cell.However, no binding of the anti-CEA binding domain to membrane-bound CEAcould be obtained.

When bispecific single chain antibodies derived from the well-describedmonoclonal antibodies T84.66 (Neumaier, M. et al., Cancer Res 50 (1990),2128-34) or MFE-23 (Boehm, M. K. Biochem J 2 (2000), 519-28) have beengenerated, these bispecific antibodies were highly sensitive to solubleCEA antigen, ie. their cytotoxic activity against CEA-positive target ortumor cells has been blocked in the presence of soluble CEA antigen.Since said constructs have been found to bind to soluble CEA, it wasconcluded that soluble CEA antigen prevents the antibody from binding tomembrane-bound CEA, thereby blocking antibody-mediated cytotoxicactivity. For example, FIG. 7 shows a cytotoxicity assay of aCEA-reactive bispecific single chain construct redirected to CHO cellstransfected with CEA in the presence of soluble human CEA. Stimulatedhuman CD8 positive cytotoxic T cells (CTLs) were used as effector cells.Cytotoxic activity of CEAII VHVL×SEQ ID NO.77 VHVL is clearly inhibitedby increasing amounts of soluble CEA. CEAII VHVL is derived from mAbT84.66; SEQ ID NO.77 is an anti-CD3 VH-VL domain.

Resistance to soluble CEA antigen could be found only for bispecificsingle chain antibodies, the CEA binding domain of which comprised theamino acid sequence “DRGLRFYFDY” of the CDR-H3 of murine monoclonalantibody A5B7 (Harwood, Br J Cancer. 54 (1986), 75-82). As for MFE-23-and T84.66-derived bispecific single chain constructs, A5B7-derivedbispecific single chain antibodies bind to soluble CEA. In light of theresults obtained for MFE-23- and T84.66-derived bispecific single chainconstructs, it could not be expected that soluble CEA does not influencecytotoxic activity in A5B7-derived single chain bispecific antibodyconstructs.

As set forth above, many therapeutic approaches directed againstCEA-bearing epithelial tumors in human are seriously hampered by thepresence of high levels of soluble CEA antigen in the plasma of patientscancer. For example, increased immune-complex formation and clearance oftherapeutic anti-CEA monoclonal antibodies in the presence of high CEAconcentrations in plasma has been observed in several clinical studies.In addition, soluble CEA antigen—frequently present in highconcentrations in the serum of cancer patients with progressive tumors,recurrent cancer, metastasic tumors, high tumor load/burden, orlate-stage tumors—blocks the therapeutics directed against CEA-positivetumor cells, thus preventing tumor cell recognition and destruction.Therefore, the actual amount of the therapeutic which reaches the tumoris reduced, resulting in a decreased, low or even no anti-tumoractivity. This limitation so far restricts e.g. antibody-basedapproaches to those patients with very low amounts of soluble CEAantigen unlikely to prevent therapeutic-tumor cell interaction.

In the present invention, it has been found that it is possible togenerate bispecific single chain antibody-therapeutics with specificityfor human CD3 and human CEA, wherein the cytotoxic activity directedagainst tumor cells is resistant to even high concentrations of solubleCEA antigen (up to 1 μg/ml soluble CEA have been tested). This findingis entirely unexpected in view of the fact that the bispecific singlechain antibodies of the invention binds to soluble CEA antigen (seeExample 3 and FIG. 2 of the present invention; see also EP B1 491031).Nevertheless, the bispecific single chain antibodies as defined hereinare entirely resistant to the presence of even high levels of solubleCEA in its cytotoxic activity towards tumor cells. Thus, the presentinvention provides means and methods particularly suited for thetreatment of tumor patients with high soluble CEA concentrations intheir plasma, as observed e.g. during tumor progression, for recurrentcancer, for metastasis, for patients with high tumor load/burden, orlate-stage tumors.

In accordance with this invention, the term “pharmaceutical composition”relates to a composition for administration to a human patient.Preferably, the pharmaceutical composition comprises suitableformulations of carriers, stabilizers and/or excipients. In a preferredembodiment, the pharmaceutical composition comprises a composition forparenteral, transdermal, intraluminal, intraarterial, intrathecal and/orintranasal administration or by direct injection into tissue. It is inparticular envisaged that said composition is administered to a patientvia infusion or injection. Administration of the suitable compositionsmay be effected by different ways, e.g., by intravenous,intraperitoneal, subcutaneous, intramuscular, topical or intradermaladministration. The composition of the present invention may furthercomprise a pharmaceutically acceptable carrier. Examples of suitablepharmaceutical carriers are well known in the art and include phosphatebuffered saline solutions, water, various types of wetting agents,sterile solutions, liposomes, etc. Compositions comprising such carrierscan be formulated by well known conventional methods. These compositionscan be administered to the subject at a suitable dose which can bedetermined e.g. by dose escalating studies by administration ofincreasing doses of the bispecific single chain antibody exhibitingresistance to soluble serum CEA antigen described herein. As set forthabove, the bispecific single chain antibody described herein withresistance to soluble serum CEA antigen can be advantageously used inthe treatment of cancer patients with high CEA serum concentrations,such as progressive tumors, recurrent cancer, metastatic tumors, hightumor load/burden, or late stage tumors. These compositions can also beadministered in combination with other proteinaceous andnon-proteinaceous drugs, e.g. in the form of a co-therapy. These drugsmay be administered simultaneously with the composition comprising thebispecific single chain antibody as defined herein or separately beforeor after administration of said bispecific antibody in timely definedintervals and doses. The dosage regimen will be determined by theattending physician and clinical factors. As is well known in themedical arts, dosages for any one patient depend upon many factors,including the patient's size, body surface area, age, the particularcompound to be administered, sex, time and route of administration,general health, and other drugs being administered concurrently.Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, and suspensions. Examples of non-aqueous solventsare propylene glycol, polyethylene glycol, vegetable oils, andinjectable organic esters such as ethyl oleate. Aqueous carriers includewater, aqueous solutions, or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, or lactated Ringer's.Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, inertgases and the like. In addition, the composition of the presentinvention might comprise proteinaceous carriers, like, e.g., serumalbumin or immunoglobulin, preferably of human origin. It is envisagedthat the co-therapy comprise, in addition to the bispecific single chainantibody as defined herein, further biologically active agents,depending on the intended use of the composition. Such agents might bedrugs acting on the gastrointestinal system, drugs acting asantineoplastic agents, chemotherapeutics, cytostatica, drugs preventinghyperurikemia, drugs inhibiting immunoreactions (e.g. corticosteroids),drugs modulating the inflammatory response, drugs acting on thecirculatory system and/or agents such as cytokines known in the art.

Preferably, the bispecific single chain antibody as defined herein isformulated in a buffer, a stabilizer and a surfactant. The buffer may bea phosphate, citrate, succinate or acetate buffer. The stabilizer may be(an) amino acid(s) and/or a sugar. The surfactants may be detergents,PEGs, or the like. More preferably, the bispecific single chain antibodyas defined herein is formulated in citrate, lysine, trehalose and Tween80. As a diluent for the pharmaceutical composition of the invention,isotonic saline and Tween 80 is preferred.

As used herein, a “bispecific single chain antibody” denotes a singlepolypeptide chain comprising two binding domains. Each “binding domain”as used herein comprises one variable region from an antibody heavychain (“VH region”), wherein the VH region of the first binding domainspecifically binds to said first molecule, i.e. the human CD3 molecule,and the VH region of the second binding domain specifically binds tohuman CEA, as defined in more detail below. The two binding domains areoptionally linked to one another by a short polypeptide spacer generallycomprising on the order of 5 amino acids. Each binding domain mayadditionally comprise one variable region from an antibody light chain(“VL region”), the VH region and VL region within each of the first andsecond binding domains being linked to one another via a polypeptidelinker, for example of the type disclosed and claimed in EP B1 623679,but in any case long enough to allow the VH region and VL region of thefirst binding domain and the VH region and VL region of the secondbinding domain to pair with one another such that, together, they areable to specifically bind to the respective first and second molecules.The arrangement of the V regions of the first or second binding domainmay be VH-VL or VL-VH. Preferably, the arrangement of the first bindingdomain specifically binding to human CD3 is VH-VL, as shown in thefollowing Examples. It is envisaged that the first binding domain may belocated N-terminally or C-terminally to the second binding domain. Thus,the arrangement of the binding domains of the bispecific single chainantibodies defined herein may be VH_(CEA)-VL_(CEA)-VH_(CD3)-VL_(CD3),VL_(CEA)-VH_(CEA)-VH_(CD3)-VL_(CD3), VH_(CD3)-VL_(CD3)-VH_(CEA)-VL_(CEA)or VH_(CD3)-VL_(CD3)-VL_(CEA)-VH_(CEA). Preferably, said first bindingdomain specific for CD3 is located C-terminally to the second bindingdomain. More preferably, the binding domains of the bispecific singlechain antibodies defined herein are arranged in the orderVH_(CEA)-VL_(CEA)-VH_(CD3)-VL_(CD3) orVL_(CEA)-VH_(CEA)-VH_(CD3)-VL_(CD3). Even more preferred, thearrangement is VL_(CEA)-VH_(CEA)-VH_(CD3)-VL_(CD3). Most preferred isthe bispecific single chain antibody construct A240 VL-B9 VH×SEQ ID NO.77 VHVL as defined in SEQ ID NO. 34. It is envisaged that said firstand/or second binding domains of the bispecific single chain antibodiesdefined herein may be of non-human origin (i.e. derived from non-humansequences). For example, said first and/or second binding domains may bederived from murine monoclonal antibodies. However, bispecific singlechain antibodies derived from murine antibodies may be recognised asforeign, when being administered to human patients. Thus, said firstand/or second binding domains of the bispecific single chain antibodiesdefined herein are preferably of human origin (i.e. derived from humansequences). Such human binding domains specifically binding to CEA orCD3 may be identified e.g. by phage display-based techniques. It is alsoenvisaged that e.g. the VH region of the first (or second) bindingdomain is a human VH region, whereas the corresponding VL region of thefirst (or second) binding domain may be of non-human origin. Suchbinding domains may be also referred to as chimeric binding domains. Orone of said binding domains is of non-human origin, whereas the other isof human origin, resulting in a chimeric bispecific single chainantibody. Said first and/or second binding domains may be furthermodified in order to reduce the immunogenicity of the bispecific singlechain antibody described herein, when being administered to humanpatients. For example, at least one of said first or second bindingdomains of the bispecific single chain antibodies defined herein may behumanized, CDR-grafted, chimeric and/or deimmunized or human, as setforth in more detail below. It is also envisaged that the polypeptidelinker linking the VH and VL region within the first and/or secondbinding domain is deimmunized. Preferably, the polypeptide linkerlinking the VH and VL region within the deimmunized first binding domain(specific for CD3) is a deimmunized polypeptide linker having thesequence “GEGTSTGS(G₂S)₂GGAD” (SEQ ID NO. 141). It is furthermoreenvisaged, that one or both of said binding domains of the bispecificsingle chain antibodies defined herein carry so-called “tags” which maybe used e.g. for protein expression, purification, detection orenrichment, such as Flag-tags, c-myc-tags, GST-tags or His-tags. Forexample, for the Flag-tag the most widely used hydrophilic octapeptidenow is DYKDDDDK (Chubet and Brizzard, Biotechniques 20 (1996):136-141)though recent studies suggest that a shorter peptide, DYKD, can berecognized with almost the same affinity by the M1 monoclonal antibody(Knappik A, Pluckthun A; Biotechniques 17 (1994):754-761). Flag-tags,c-myc-tags, GST-tags, His-tags or the like may be positioned either atthe N-terminus or the C-terminus of the bispecific single chainantibodies such as, for instance,tag-VH_(CEA)-VL_(CEA)-VH_(CD3)-VL_(CD3) orVL_(CEA)-VH_(CEA)-VH_(CD3)-VL_(CD3)-tag. The sources for and propertiesof such tags for expression, detection or purification purposes are welldescribed in the art; see e.g. Lichty, Protein Expr Purif. 41 (2005),98-105.

As used herein, the term “single-chain Fv” or “scFv” refers to antibodyfragments comprising the VH and VL domains of an antibody, wherein thesedomains are present in a single polypeptide chain. Variable domains canbe arranged in the order VH-VL or VL-VH. Generally, the Fv polypeptidefurther comprises a polypeptide linker between the VH and VL domainswhich enables the scFv to form the desired structure for antigenbinding. For a review of scFv see Pluckthun in The Pharmacology ofMonoclonal Antibodies, vol. 113, Rosenburg and Moore eds.Springer-Verlag, New York, pp. 269-315 (1994). In a specific embodiment,the invention relates to anti-CEA scFvs derived from the bispecificsingle chain antibodies defined herein.

According to the present invention, the term “binding domain” or“variable region” used in the context with Ig-derivedantigen-interaction comprises fragments and derivatives of polypeptideswhich at least comprise one CDR derived from an antibody, antibodyfragment or derivative thereof. It is envisaged by the invention, thatthe second binding domain specifically binding to human CEA of thebispecific single chain antibody defined herein comprises at least oneCDR, preferably a CDR-H3, more preferably a part of the CDR-H3 of murinemonoclonal antibody A5B7 with the amino acid sequence “FYFDY” (SEQ IDNO. 112) corresponding to Kabat positions 100, 100a, 100b, 101, and 102,respectively, of CDR-H3 of murine monoclonal antibody A5B7; even morepreferred with the amino acid sequence “DX₁X₂X₃X₄FYFDY” (SEQ ID NO. 65),wherein “X₁”, “X₂”, “X₃” or “X₄” represents any amino acid residue, andthe amino acid residue “D” corresponds to Kabat position 95 of CDR-H3 ofmurine monoclonal antibody A5B7 and the amino acid residues “FYFDY”correspond to Kabat positions 100, 100a, 100b, 101, and 102,respectively, of CDR-H3 of murine monoclonal antibody A5B7. It isenvisaged that “X₁”, “X₂”, “X₃” or “X₄” corresponding to Kabat positions96 (“X₁”), 97 (“X₂”), 98 (“X₃”) and 99 (“X₄”), respectively, of CDR-H3of murine monoclonal antibody A5B7, represent amino acid residue “R”(Arginine), “G” (Glycine), “L” (Leucine), “Y” (Tyrosine), “A” (Alanine),“D” (Aspartic acid), “S” (Serine), “W” (Tryptophan), “F” (Phenylalanine)or “T” (Threonine). Herein, it is excluded from the scope of theinvention that “X₁”, “X₂”, “X₃” and “X₄” represent the same amino acid,e.g. that “X₁”, “X₂”, “X₃” and “X₄” are all “F” (Phenylalanine).Preferably, “X₁” represents “R” (Arginine), “F” (Phenylalanine), “M”(Methionine), “E” (Glutamic acid), or “T” (Threonine); “X₂” represents“G” (Glycine), “Y” (Tyrosine), “A” (Alanine), “D” (Aspartic acid), or“S” (Serine); “X₃” represents “L” (Leucine), “F” (Phenylalanine), “M”(Methionine), “E” (Glutamic acid), or “T” (Threonine); and “X₄”represents “R” (Arginine), “Y” (Tyrosine), “A” (Alanine), “D” (Asparticacid), or “S” (Serine). Or most preferred the second binding domainspecifically binding to human CEA of the bispecific single chainantibody defined herein comprises the complete CDR-H3 of A5B7 with theamino acid sequence “DRGLRFYFDY” (SEQ ID NO. 66) corresponding to Kabatpositions 95-102 of the CDR-H3 of A5B7. As shown in the followingExamples, the cytotoxic activity against tumor cells of the bispecificsingle chain antibody defined herein comprising said mAb A5B7-derivedCDR-H3 “DRGLRFYFDY” (SEQ ID NO. 66) amino acid sequence in the secondbinding domain interacting with CEA are resistant to soluble CEAantigen, thereby allowing the treatment of tumor patients with highserum CEA concentrations in their plasma. Determination of CDRs is knownto the person skilled in the art; see e.g.http://www.bioinf.org.uk/abs/#cdrid. Numbering of amino acid sequencesin antibodies can be carried out e.g. according to the Kabat numberingscheme described in the art; see e.g. Kabat, E. A., T. T. Wu, H. M.Perry, K. S. Gottesman, and C. Foeller. 1991. Sequences of Proteins ofImmunological Interest, 5th ed. Bethesda, Md.: National Center forBiotechnology Information, National Library of Medicine.

Most preferably and as documented in the appended examples, the“bispecific single chain antibody” to be employed in the pharmaceuticalcomposition of the invention is a bispecific single chain Fv (scFv) witha deimmunized anti-CD3 binding domain (WO 2005/040220) and a humananti-CEA binding domain comprising at least the amino acid sequence“DRGLRFYFDY” corresponding to Kabat positions 95-102 (SEQ ID NO. 66) ofthe CDR-H3 of murine monoclonal antibody A5B7. Bispecific single chainmolecules are known in the art and are described e.g. in WO 99/54440 orMack, PNAS, (1995), 92, 7021-7025.

The term “single-chain” as used in accordance with the present inventionmeans that said first and second domain of the bispecific single chainconstruct are covalently linked, preferably in the form of a co-linearamino acid sequence encodable by a single nucleic acid molecule.

As used herein, “human” refers to the species Homo sapiens. A “human”molecule, e.g. human CEA or human CD3 (CD3 epsilon), is therefore thevariant of that molecule as it is naturally expressed in Homo sapiens.

The term “epithelial tumor” as used herein denotes a tumor of epithelialorigin which is CEA positive (Cancer Medicine; 6th ed.; Kufe, Donald W.;Pollock, Raphael E.; Weichselbaum, Ralph R.; Bast, Robert C., Jr.;Gansler, Ted S.; Holland, James F.; Frei III, Emil, editors. Hamilton(Canada): BC Decker Inc. 2003; http://www.dkfz.de;http://www.krebsinformationsdienst.de/Krebsarten/index.html). Theepithelial tumor to be treated may be a gastrointestinal adenocarcinoma,a breast adenocarcinoma or a lung adenocarcinoma. Said gastrointestinaladenocarcinoma is preferably a colorectal, pancreatic, an oesophageal ora gastric adenocarcinoma. As set forth herein, the pharmaceuticalcomposition of the invention is particularly advantageous for thetreatment of patients with progressive tumors, metastasis, recurrentcancer, late stage epithelial tumors, high epithelial tumor load/tumorburden, or tumor patients with a CEA serum concentration higher than 100ng/ml (as determined e.g. by ELISA), characterized by high levels ofsoluble CEA antigen in the plasma of the tumor patients. It is alsowithin the scope of the invention that said pharmaceutical compositionbe used after surgical removal of the primary tumor. For example,disseminated residual tumor cells derived from a CEA producingepithelial tumor also shed CEA into their microenvironments. Thus, inthe surrounding of these tumor cells the level of soluble CEA is alsohigh. Accordingly, resistance to soluble CEA of cytotoxic activity ofpharmaceutical compositions of the invention is advantageous also forthe treatment of minimal residual disease. It is envisaged that saidpharmaceutical compositions may be administered in a period in whichserum CEA levels decrease (due to the removal of the CEA source, i.e.the primary tumor) in order to kill remaining tumor cells. Thepharmaceutical compositions may also be useful after the removal of theprimary tumor, in the case that serum CEA levels increase due to theformation of secondary tumors or metastasis. The CEA serum concentrationcan be determined e.g. by CEA ELISA assays (see e.g. IBL CEA EIA, IBLHamburg, Germany). As set forth above, in many antibody-basedtherapeutic approaches, said serum CEA inhibits binding of the antibodyto membrane-bound CEA on the tumor cells and blocks the activity ofantibody, thereby worsening the success of the anti-tumor therapy.

As used herein, the term “specifically binds” or related expressionssuch as “specifically binding” or “specific reactivity with/to” etc.refer to the ability of the first and/or second binding domains of thebispecific single chain antibody as defined herein to discriminatebetween the respective first and/or second molecule to such an extentthat, from a pool of a plurality of different molecules as potentialbinding partners, only said respective first and/or second moleculeis/are bound, or is/are significantly bound. Such binding measurementscan be routinely performed e.g. on a Biacore apparatus, by ELISA, FACSanalysis or the like. More specifically, the first binding domain of thebispecific single chain antibody as defined herein binds to human CD3,preferably human CD3 epsilon. The second binding domain of thebispecific single chain antibodies as defined herein binds to aepithelial tumor antigen, i.e. human CEA (carcinoembryonic antigen,carcinoembryonic antigen-related cell adhesion molecule 5; CEACAM5;CD66e), as set forth below. The term “specifically binding” means inaccordance with this invention that the bispecific single chain antibodymolecule is capable of specifically interacting with and/or binding toat least two, three, four, five, six, seven, eight or even more aminoacids of each of the human target molecule as defined herein. Said termrelates to the specificity of the antibody molecule, i.e. to its abilityto discriminate between the specific regions of the human targetmolecule as defined herein. The specific interaction of theantigen-interaction-site with its specific antigen may result in aninitiation of a signal, e.g. due to the induction of a change of theconformation of the antigen, an oligomerization of the antigen, etc.Further, said binding may be exemplified by the specificity of a“key-lock-principle”. Thus, specific motifs in the amino acid sequenceof the antigen-interaction-site and the antigen bind to each other as aresult of their primary, secondary or tertiary structure as well as theresult of secondary modifications of said structure. The specificinteraction of the antigen-interaction-site with its specific antigenmay result as well in a binding of said site to the antigen.

The “specific binding” of an antibody is characterized primarily by twoparameters: a qualitative parameter (the binding epitope, or where theantibody binds) and a quantitative parameter (the binding affinity, orhow strongly it binds where it does). Which epitope is bound by anantibody can advantageously be determined by e.g. known FACSmethodology, peptide-spot epitope mapping, mass spectroscopy or peptideELISA. The strength of antibody binding to a particular epitope may beadvantageously be determined by e.g. known Biacore and/or ELISAmethodologies. A combination of such techniques allows the calculationof a signal:noise ratio as a representative measure of bindingspecificity. In such a signal:noise ratio, the signal represents thestrength of antibody binding to the epitope of interest, whereas thenoise represents the strength of antibody binding to other, non-relatedepitopes differing from the epitope of interest. Preferably, asignal:noise ratio for an epitope of interest which is about 50-foldhigher than for other epitopes different from the epitope of interestmay be taken as an indication that the antibody evaluated binds theepitope of interest in a specific manner, i.e. is a “specific binder”.

The term “specific binding” or “specific interaction” as used inaccordance with the present invention means that the bispecific singlechain construct does not or essentially does not cross-react withpolypeptides of similar structures. Cross-reactivity of a panel ofbispecific single chain construct under investigation may be tested, forexample, by assessing binding of said panel of bispecific single chainconstruct under conventional conditions (see, e.g., Harlow and Lane,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,1988 and Using Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory Press, 1999) to the polypeptide of interest as well as to anumber of more or less (structurally and/or functionally) closelyrelated polypeptides. For example, it is within the scope of theinvention that the first binding domain of the bispecific single chainantibody of the invention binds to human CEA (carcinoembryonic antigen;CEACAM5; CEA; CD66e) i.e. both to soluble CEA antigen and tomembrane-bound CEA, whereas bispecific antibodies binding to other CEAfamily members, such as biliary glycoprotein (CEACAM1; BGP1; TM-CEA;CD66a), are excluded from said scope.

Examples for the specific interaction of an antigen-interaction-sitewith a specific antigen comprise the specificity of a ligand for itsreceptor. Said definition particularly comprises the interaction ofligands which induce a signal upon binding to its specific receptor.Examples for corresponding ligands comprise cytokines whichinteract/bind with/to its specific cytokine-receptors. Also particularlycomprised by said definition is the binding of anantigen-interaction-site to antigens like antigens of the selectinfamily, integrins and of the family of growth factors like EGF. Anotherexample for said interaction, which is also particularly comprised bysaid definition, is the interaction of an antigenic determinant(epitope) with the antigenic binding site of an antibody.

The term “binding to/interacting with” may also relate to aconformational epitope, a structural epitope or a discontinuous epitopeconsisting of two regions of the human target molecules or partsthereof. In context of this invention, a conformational epitope isdefined by two or more discrete amino acid sequences separated in theprimary sequence which come together on the surface of the molecule whenthe polypeptide folds to the native protein (Sela, (1969) Science 166,1365 and Laver, (1990) Cell 61, 553-6).

The term “discontinuous epitope” means in context of the inventionnon-linear epitopes that are assembled from residues from distantportions of the polypeptide chain. These residues come together on thesurface of the molecule when the polypeptide chain folds into athree-dimensional structure to constitute a conformational/structuralepitope.

“CD3” as used herein denotes an antigen that is expressed on T-cells aspart of the multimolecular T-cell receptor complex and that consists ofat least five different chains, CD3-gamma, -delta, -epsilon, -zeta, and-eta. Clustering of CD3 on T-cells, e.g., by immobilizedanti-CD3-antibodies, leads to T-cell activation similar to theengagement of the T-cell receptor but independent from its clone typicalspecificity. Actually, most anti-CD3-antibodies recognize the CD3epsilon-chain. The amino acid sequence of human CD3 epsilon is depictedin GenBank accession number NM_000733 and comprises SEQ ID NO. 111.

“CEA” denotes the carcinoembryonic antigen (carcinoembryonicantigen-related cell adhesion molecule 5; CEACAM5; CEA; CD66e), anantigen expressed in a large number of tumors of epithelial origin(Hammarström, Sem. Cancer Biol. 9 (1999), 67-81; Shively and Beatty CRCCrit. Rev. Oncol. Hematol. 2 (1985), 355-399). The amino acid sequenceof human CEA is depicted in GenBank accession number NM_004363 andcomprises SEQ ID NO. 76.

In the present invention, it has been surprisingly found that it ispossible to generate antibody-based therapeutics with specificity forhuman CD3 and human CEA, wherein the cytotoxic activity directed againsttumor cells is resistant to even high concentrations of soluble CEAantigen. This finding is entirely unexpected in view of the fact thatthe bispecific single chain antibodies of the invention bind to solubleCEA antigen. For example, when bispecific single chain antibodyconstructs derived from monoclonal antibodies T84.66 or MFE-23 have beengenerated, these antibodies were highly sensitive to soluble CEAantigen, i.e. their cytotoxic activity has been blocked in the presenceof soluble CEA antigen. The inhibition of the cytotoxic activity of saidconstructs by soluble CEA could also not be overcome by increasedamounts of antibody. These constructs have also been found to be capableof binding to soluble CEA. In view of this, it was concluded thatsoluble CEA antigen prevents the antibody from exerting its cytotoxicactivity. In contrast, the bispecific single chain antibodies as definedherein are entirely resistant to the presence of even high levels ofsoluble CEA in their cytotoxic activity towards tumor cells. Moreover,due to their high cytotoxic activity, said bispecific constructs asdefined herein elicit their biological activity at even lowconcentrations. Hence, low amounts of pharmaceutical compositionscomprising the bispecific single chain antibodies as defined herein aresufficient to achieve a therapeutic effect in epithelial tumor patientscharacterized by high soluble CEA concentrations in their serum/plasma.High soluble CEA concentrations in the serum/plasma of epithelial tumorpatients are characteristic for progressive, recurrent, metastatic, orlate stage tumors and for patients with high tumor load. Even moresurprising, it has been found that the amino acid sequence “DRGLRFYFDY”(SEQ ID NO. 66) corresponding to Kabat positions 95-102 of the CDR-H3 ofmurine monoclonal antibody A5B7 is sufficient to mediate resistance tosoluble CEA antigen when used in a human CEA-binding domain (i.e. humanbinding domains specifically binding to human CEA) of anti-CEAxanti-CD3bispecific single chain antibodies. Due to their human origin, saidconstructs are low or non-immunogenic when being administered to humantumor patients. In summary, the pharmaceutical compositions comprisingthe bispecific single chain antibodies as defined herein areparticularly useful for the treatment of epithelial tumor patients withhigh soluble CEA concentrations in their plasma, as observed e.g. duringtumor progression, for recurrent cancer, for metastasis, for patientswith high tumor load/burden, or late-stage tumors.

In another preferred embodiment of the pharmaceutical composition of theinvention, said first binding domain specific for CD3 of the bispecificsingle chain antibodies defined herein is located C-terminally to thesecond binding domain.

Within the scope of the invention and all embodiments thereof, the orderof arrangement of the first and second binding domains on the singlepolypeptide chain, i.e. within the bispecific single chain antibodydefined herein, is relevant. It is envisaged that the arrangement of thebinding domains of the bispecific single chain antibodies defined hereinmay be VH_(CEA)-VL_(CEA)-VH_(CD3)-VL_(CD3),VL_(CEA)-VH_(CEA)-VH_(CD3)-VL_(CD3), VH_(CD3)-VL_(CD3)-VH_(CEA)-VL_(CEA)or VH_(CD3)-VL_(CD3)-VL_(CEA)-VH_(CEA). As shown in the followingexamples, the advantages as described hereinabove are particularlyrealizable when the first binding domain (specifically binding to CD3)is located C-terminally to the second binding domain, i.e. closer to theC-terminus of the bispecific single chain antibody than the secondbinding domain. It is preferred that the first binding domainspecifically binding to human CD3 is arranged in the VH-VL orientation.For example, the binding domains of the bispecific single chainantibodies defined herein may be arranged in the orderVH_(CEA)-VL_(CEA)-VH_(CD3)-VL_(CD3) orVL_(CEA)-VH_(CEA)-VH_(CD3)-VL_(CD3). As used herein, “N-terminally to”or “C-terminally to” and grammatical variants thereof denote relativelocation within the primary amino acid sequence rather than placement atthe absolute N- or C-terminus of the bispecific single chain antibody.Hence, as a non-limiting example, a first binding domain which is“located C-terminally to the second binding domain” simply denotes thatthe first binding domain is located to the carboxyl side of the secondbinding domain within the bispecific single chain antibody, and does notexclude the possibility that an additional sequence, for example a tagas set forth above, or another proteinaceous or non-proteinaceouscompound such as a radioisotope, is located at the ultimate C-terminusof the bispecific single chain antibody.

Preferably, said binding domains of the bispecific single chainantibodies defined herein are arranged in the orderVH_(CEA)-VL_(CEA)-VH_(CD3)-VL_(CD3) orVL_(CEA)-VH_(CEA)-VH_(CD3)-VL_(CD3). Even more preferred, thearrangement is VL_(CEA)-VH_(CEA)-VH_(CD3)-VL_(CD3). Most preferred isthe bispecific single chain antibody construct A240 VL-B9 VH×SEQ ID NO.77 VHVL as defined in SEQ ID NO. 34.

It is preferred that the second binding domain specifically binding tohuman CEA of the bispecific single chain antibody defined hereincomprises at least one CDR, preferably a CDR-H3, more preferably a partof the CDR-H3 of murine monoclonal antibody A5B7 with the amino acidsequence “FYFDY” (SEQ ID NO. 112) corresponding to Kabat positions 100,100a, 100b, 101, and 102, respectively, of CDR-H3 of murine monoclonalantibody A5B7; even more preferred with the amino acid sequence“DX₁X₂X₃X₄FYFDY” (SEQ ID NO. 65), wherein “X₁”, “X₂”, “X₃” or “X₄”represents any amino acid residue, and the amino acid residue “D”corresponds to Kabat position 95 of CDR-H3 of murine monoclonal antibodyA5B7 and the amino acid residues “FYFDY” correspond to Kabat positions100, 100a, 100b, 101, and 102, respectively, of CDR-H3 of murinemonoclonal antibody A5B7. Herein, “X₁”, “X₂”, “X₃” and “X₄” correspondto Kabat positions 96 (“X₁”), 97 (“X₂”), 98 (“X₃”) and 99 (“X₄”),respectively, of CDR-H3 of murine monoclonal antibody A5B7. It isenvisaged that “X₁”, “X₂”, “X₃” or “X₄” represent amino acid residue “R”(Arginine), “G” (Glycine), “L” (Leucine), “Y” (Tyrosine), “A” (Alanine),“D” (Aspartic acid), “S” (Serine), “W” (Tryptophan), “F” (Phenylalanine)or “T” (Threonine). Herein, it is excluded from the scope of the claimsof the invention that “X₁”, “X₂”, “X₃” and “X₄” represent the same aminoacid, e.g. that “X₁”, “X₂”, “X₃” and “X₄” are all “F” (Phenylalanine).Preferably, “X₁” represents “R” (Arginine), “F” (Phenylalanine), “M”(Methionine), “E” (Glutamic acid), or “T” (Threonine); “X₂” represents“G” (Glycine), “Y” (Tyrosine), “A” (Alanine), “D” (Aspartic acid), or“S” (Serine); “X₃” represents “L” (Leucine), “F” (Phenylalanine), “M”(Methionine), “E” (Glutamic acid), or “T” (Threonine); and “X₄”represents “R” (Arginine), “Y” (Tyrosine), “A” (Alanine), “D” (Asparticacid), or “S” (Serine). Even more preferred, the second binding domainspecific for human CEA comprises at least the amino acid sequence“RFYFDY” (SEQ ID NO. 113), “LRFYFDY” (SEQ ID NO. 114), “GLRFYFDY” (SEQID NO. 115), or “RGLRFYFDY” (SEQ ID NO. 116) of CDR-H3 of monoclonalantibody A5B7. Most preferred is the complete CDR-H3 of A5B7 with theamino acid sequence “DRGLRFYFDY” (SEQ ID NO. 66) corresponding to Kabatpositions 95 (“D”, Aspartic acid), 96 (“R”; Arginine), 97 (“G”;Glycine), 98 (“L”; Leucine), 99 (“R”; Arginine), 100 (“F”;Phenylalanine), 100a (“Y”; Tyrosine), 100b (“F”; Phenylalanine), 101(“D”; Aspartic acid), and 102 (“Y”; Tyrosine), respectively. Numberingaccording to the Kabat system is set forth e.g. in Kabat, E. A., T. T.Wu, H. M. Perry, K. S. Gottesman, and C. Foeller. 1991. Sequences ofProteins of Immunological Interest, 5th ed. Bethesda, Md.: NationalCenter for Biotechnology Information, National Library of Medicine.

As shown in the following Examples, the cytotoxic activity against tumorcells of the bispecific single chain antibody defined herein comprisingsaid mAb A5B7-derived CDR-H3 “DRGLRFYFDY” (SEQ ID NO. 66) amino acidsequence in the second binding domain interacting with CEA are resistantto soluble CEA antigen, thereby allowing the treatment of tumor patientswith high serum CEA concentrations in their plasma.

It may be desirable to further modify this A5B7-derived “DRGLRFYFDY”CDR-H3 amino acid sequence e.g. in order to improve affinity for the CEAtarget antigen (on the epithelial tumor cells) and/or to optimize “finespecificity” of the bispecific single chain antibody as defined herein.To this end, in the amino acid sequence “DX₁X₂X₃X₄FYFDY” (SEQ ID NO.65)”, various amino acid residues may be tested at positions “X₁”, “X₂”,“X₃” and/or “X₄” (corresponding to Kabat positions 96 (“X₁”), 97 (“X₂”),98 (“X₃”) and 99 (“X₄”), respectively, of CDR-H3 of murine monoclonalantibody A5B7) in order to identify a modified CDR-H3 with improvedaffinity and/or fine specificity. For instance, “X₁”, “X₂”, “X₃” or “X₄”may represent amino acid residue “R” (Arginine), “G” (Glycine), “L”(Leucine), “Y” (Tyrosine), “A” (Alanine), “D” (Aspartic acid), “S”(Serine), “W” (Tryptophan), “F” (Phenylalanine) or “T” (Threonine).Herein, one, two, three or all four of the indicated “X” positions maybe exchanged in comparison to the original “RGLR” amino acid sequence atKabat positions 96 to 99 in the CDR-H3 “DRGLRFYFDY” (SEQ ID NO. 66)amino acid sequence. However, it is excluded from the scope of theclaims of the invention that “X₁”, “X₂”, “X₃” and “X₄” represent thesame amino acid, e.g. that “X₁”, “X₂”, “X₃” and “X₄” are all “F”(Phenylalanine). The above-mentioned modification of the A5B7-derived“DRGLRFYFDY” CDR-H3 amino acid sequence can be achieved by methods knownin the art, such as PCR using randomized primers, which allows thegeneration of bispecific single chain antibodies with such modifiedCDR-H3 regions in the CEA-binding domain. Affinity or fine specificityof these modified bispecific single chain antibodies can be tested bymethods described in the art, e.g. by ELISA, Biacore or FACS analysis.The resistance to soluble CEA antigen of a bispecific single chainantibody with such a modified CDR-H3 can be tested in cytotoxicityassays in the presence of increasing amounts of soluble CEA, asdescribed in the following Examples.

More preferably, said second binding domain specific for human CEA ofthe bispecific single chain antibodies defined herein comprises SEQ IDNO. 65 or 66 and/or a CDR-H1 having the amino acid sequence “SYWMH” (SEQID NO. 68) and/or a CDR-H2 having the amino acid sequence“FIRNKANGGTTEYAASVKG” (SEQ ID NO. 67) or “FILNKANGGTTEYAASVKG” (SEQ IDNO. 145). Thus, said second binding domain specific for human CEA of thebispecific single chain antibodies defined herein may comprise one, twoor three CDR-H regions as defined above. Alternatively, said secondbinding domain specific for human CEA of the bispecific single chainantibodies defined herein comprises SEQ ID NO. 65 or 66 and/or a CDR-H1having the amino acid sequence “TYAMH” (SEQ ID NO. 70) and/or a CDR-H2having the amino acid sequence “LISNDGSNKYYADSVKG” (SEQ ID NO. 69).Thus, alternatively, said second binding domain specific for human CEAof the bispecific single chain antibodies defined herein may compriseone, two or three CDR-H regions as defined above. Even more preferred,said second binding domain specific for human CEA of the bispecificsingle chain antibodies defined herein in addition to the one, two orthree CDR-H regions as depicted above comprises a CDR-L1 having theamino acid sequence “TLRRGINVGAYSIY” (SEQ ID NO. 73) and/or a CDR-L2having the amino acid sequence “YKSDSDKQQGS” (SEQ ID NO. 72) and/or aCDR-L3 having the amino acid sequence “MIWHSGASAV” (SEQ ID NO. 71).

The amino acid sequence of the VH region of the second binding domainspecific for human CEA of the bispecific single chain antibodies definedherein is preferably SEQ ID NO. 60 comprising “DRGLRFYFDY” (SEQ ID NO.66) corresponding to Kabat positions 95-102 of the CDR-H3 of murinemonoclonal antibody A5B7 and a CDR-H1 having the amino acid sequence“SYWMH” (SEQ ID NO. 68) and a CDR-H2 having the amino acid sequence“FIRNKANGGTTEYAASVKG” (SEQ ID NO. 67).

The amino acid sequence of the VH region of the second binding domainspecific for human CEA of the bispecific single chain antibodies definedherein is preferably SEQ ID NO. 146 comprising “DRGLRFYFDY” (SEQ ID NO.66) corresponding to Kabat positions 95-102 of the CDR-H3 of murinemonoclonal antibody A5B7 and a CDR-H1 having the amino acid sequence“SYWMH” (SEQ ID NO. 68) and a CDR-H2 having the amino acid sequence“FILNKANGGTTEYAASVKG” (SEQ ID NO. 145).

The amino acid sequence of the VH region of the second binding domainspecific for human CEA of the bispecific single chain antibodies definedherein is preferably SEQ

ID NO. 58 or SEQ ID NO. 62 comprising “DRGLRFYFDY” (SEQ ID NO. 66)corresponding to Kabat positions 95-102 of the CDR-H3 of murinemonoclonal antibody A5B7 and a CDR-H1 having the amino acid sequence“TYAMH” (SEQ ID NO. 70) and a CDR-H2 having the amino acid sequence“LISNDGSNKYYADSVKG” (SEQ ID NO. 69).

The VL region of the second binding domain specific for human CEA of thebispecific single chain antibodies defined herein is preferably SEQ IDNO. 64 comprising CDR-L1 having the amino acid sequence “TLRRGINVGAYSIY”(SEQ ID NO. 73) and a CDR-L2 having the amino acid sequence“YKSDSDKQQGS” (SEQ ID NO. 72) and a CDR-L3 having the amino acidsequence “MIWHSGASAV” (SEQ ID NO. 71).

As set forth above, the order or arrangement of the variable regions ofthe second binding domain specifically binding to CEA may be VH-VL orVL-VH. Both arrangements are within the scope of the invention. For asecond binding domain comprising the VH of SEQ ID NO. 60 and the VL ofSEQ ID NO. 64, the VH-VL arrangement is shown in SEQ ID NO. 52, whereasthe VL-VH arrangement is depicted in SEQ ID NO. 122. For a secondbinding domain comprising the VH of SEQ ID NO. 146 and the VL of SEQ IDNO. 64, the VH-VL arrangement is shown in SEQ ID NO. 147.

For a secondbinding domain comprising the VH of SEQ ID NO. 58 and the VLof SEQ ID NO. 64, the VH-VL arrangement is shown in SEQ ID NO 50,whereas the VL-VH arrangement is shown in SEQ ID NO. 120. For asecondbinding domain comprising the VH of SEQ ID NO. 62 and the VL ofSEQ ID NO. 64, the VH-VL arrangement is shown in SEQ ID NO. 54, whereasthe VL-VH arrangement is depicted in SEQ ID NO. 124. For a secondbindingdomain comprising the VH of SEQ ID NO. 56 and the VL of SEQ ID NO. 64,the VH-VL arrangement is shown in SEQ ID NO. 48, whereas the VL-VHarrangement is depicted in SEQ ID NO. 118.

Even more preferred, the V regions of the second binding domain specificfor CEA of the bispecific single chain antibodies defined herein areselected from the group consisting of:

(a) the VH region consists of the amino acid sequence shown in SEQ IDNO. 60 and the VL region consists of the amino acid sequence shown inSEQ ID NO. 64;

(b) the VH region consists of the amino acid sequence shown in SEQ IDNO. 146 and the VL region consists of the amino acid sequence shown inSEQ ID NO. 64;

(c) the VH region consists of the amino acid sequence shown in SEQ IDNO. 58 and the VL region consists of the amino acid sequence shown inSEQ ID NO. 64;

(d) the VH region consists of the amino acid sequence shown in SEQ IDNO. 62 and the VL region consists of the amino acid sequence shown inSEQ ID NO. 64; and

(e) the VH region consists of the amino acid sequence shown in SEQ IDNO. 56 and the VL region consists of the amino acid sequence shown inSEQ ID NO. 64.

Most preferred, said bispecific single chain antibody comprises an aminoacid sequence selected from the group consisting of:

-   -   (a) an amino acid sequence as depicted in any of SEQ ID NOs. 6,        8, 16, 18, 24, 26, 32, 34, 40, 42, 126, 130, 134 or 143;    -   (b) an amino acid sequence encoded by a nucleic acid sequence as        shown in SEQ ID NOs. 5, 7, 15, 17, 23, 25, 31, 33, 39, 41, 125,        129, 133 or 142;    -   (c) an amino acid sequence encoded by a nucleic acid sequence        hybridising under stringent conditions to the complementary        nucleic acid sequence of (b);    -   (d) an amino acid sequence encoded by a nucleic acid sequence        which is degenerate as a result of the genetic code to a        nucleotide sequence of (b); and    -   (e) an amino acid sequence at least 85% identical, more        preferred at least 90% identical, most preferred at least 95%        identical to the amino acid sequence of (a) or (b).

In another preferred embodiment of the pharmaceutical composition of theinvention, said epithelial tumor to be treated is a gastrointestinaladenocarcinoma, a breast adenocarcinoma or a lung adenocarcinoma. Saidgastrointestinal adenocarcinoma is preferably a colorectal, pancreatic,an oesophageal or a gastric adenocarcinoma.

More preferably, said pharmaceutical composition of the invention is forthe treatment of progressive tumors, late stage tumors, tumor patientswith high tumor load/burden, metastatic tumors, or tumor patients with aCEA serum concentration higher than 100 ng/ml. Said CEA serumconcentration may be determined e.g. by ELISA.

In a further preferred embodiment of the pharmaceutical composition ofthe invention, at least one of said first or second binding domains ofthe bispecific single chain antibodies defined herein is chimeric,humanized, CDR-grafted, and/or deimmunized or human.

The term “chimeric” as used herein has been defined above. The term“human” binding domain, e.g. a human binding domain specifically bindingto human CEA as used herein is to be understood as meaning that thebispecific single chain antibody as defined herein comprises (an) aminoacid sequence(s) contained in the human germline antibody repertoire orantibody repertoire having at least the amino acid sequence “FYFDY”corresponding to Kabat positions 100, 100a, 100b, 101, and 102 (SEQ IDNO. 112) of the CDR-H3 of murine monoclonal antibody A5B7 or aA5B7-derived CDR-H3 as defined above. A bispecific single chain antibodyas defined herein may also be regarded as human if it consists of (a)sequence(s) that deviate(s) from its (their) closest human germlinesequence(s) by no more than would be expected due to the imprint ofsomatic hypermutation. Additionally, the antibodies of many non-humanmammals, for example rodents such as mice and rats, comprise VH CDRamino acid sequences which one may expect to exist in the expressedhuman antibody repertoire as well. Any such sequence(s) of human ornon-human origin which may be expected to exist in the expressed humanrepertoire would also be considered “human” for the purposes of thepresent invention.

As used herein, the term “humanized”, “humanization” or “human-like” areused interchangeably to refer to a bispecific single chain antibodycomprising in at least one of its binding domains at least onecomplementarity determining region (“CDR”) from a non-human antibody orfragment thereof. Humanization approaches are described for example inWO 91/09968 and U.S. Pat. No. 6,407,213. As non-limiting examples, theterm encompasses the case in which a variable region of at least onebinding domain comprises a single CDR region, for example the third CDRregion of the VH, from another non-human animal, for example a rodent,as well as the case in which a or both variable region/s comprise ateach of their respective first, second and third CDRs the CDRs from saidnon-human animal. In the event that all CDRs of a binding domain of thebispecific single chain antibody have been replaced by theircorresponding equivalents from, for example, a rodent, one typicallyspeaks of “CDR-grafting”, and this term is to be understood as beingencompassed by the term “humanized” or grammatically related variantsthereof as used herein. The term “humanized” or grammatically relatedvariants thereof also encompasses cases in which, in addition toreplacement of one or more CDR regions within a VH and/or VL of thefirst and/or second binding domain further mutation/s (e.g.substitutions) of at least one single amino acid residue/s within theframework (“FR”) regions between the CDRs has/have been effected suchthat the amino acids at that/those positions correspond/s to the aminoacid/s at that/those position/s in the animal from which the CDR regionsused for replacement is/are derived. As is known in the art, suchindividual mutations are often made in the framework regions followingCDR-grafting in order to restore the original binding affinity of thenon-human antibody used as a CDR-donor for its target molecule. The term“humanized” may further encompass (an) amino acid substitution(s) in theCDR regions from a non-human animal to the amino acid(s) of acorresponding CDR region from a human antibody, in addition to the aminoacid substitutions in the framework regions as described above.

As used herein, the term “deimmunized” or “deimmunization” denotesmodification of the first and/or second binding domain vis-à-vis anoriginal wild type construct by rendering said wild type constructnon-immunogenic or less immunogenic in humans. Deimmunization approachesare shown e.g. in WO 00/34317, WO 98/52976, WO 02/079415 or WO 92/10755.The term “deimmunized” also relates to constructs, which show reducedpropensity to generate T cell epitopes. In accordance with thisinvention, the term “reduced propensity to generate T cell epitopes”relates to the removal of T-cell epitopes leading to specific T-cellactivation. Furthermore, “reduced propensity to generate T cellepitopes” means substitution of amino acids contributing to theformation of T cell epitopes, i.e. substitution of amino acids, whichare essential for formation of a T cell epitope. In other words,“reduced propensity to generate T cell epitopes” relates to reducedimmunogenicity or reduced capacity to induce antigen independent T cellproliferation. The term “T cell epitope” relates to short peptidesequences which can be released during the degradation of peptides,polypeptides or proteins within cells and subsequently be presented bymolecules of the major histocompatibility complex (MHC) in order totrigger the activation of T cells; see inter alia WO 02/066514. Forpeptides presented by MHC class II such activation of T cells can thengive rise to an antibody response by direct stimulation of T cells toproduce said antibodies. “Reduced propensity to generate T-cellepitopes” and/or “deimmunization” may be measured by techniques known inthe art. Preferably, de-immunization of proteins may be tested in vitroby T cell proliferation assay. In this assay PBMCs from donorsrepresenting >80% of HLA-DR alleles in the world are screened forproliferation in response to either wild type or de-immunized peptides.Ideally cell proliferation is only detected upon loading of theantigen-presenting cells with wild type peptides. Alternatively, one maytest deimmunization by expressing HLA-DR tetramers representing allhaplotypes. These tetramers may be tested for peptide binding or loadedwith peptides substitute for antigen-presenting cells in proliferationassays. In order to test whether deimmunized peptides are presented onHLA-DR haplotypes, binding of e.g. fluorescence-labeled peptides onPBMCs can be measured. Furthermore, deimmunization can be proven bydetermining whether antibodies against the deimmunized molecules havebeen formed after administration in patients. Preferably, antibodyderived molecules are deimmunized in the framework regions and most ofthe CDR regions are not modified in order to generate reduced propensityto induce T cell epitope so that the binding affinity of the CDR regionsis not affected. Even elimination of one T cell epitope results inreduced immunogenicity.

In summary, the above approaches help to reduce the immunogenicity ofthe therapeutic bispecific single chain antibodies as defined hereinwhen being administered to epithelial tumor patients. For example, thefirst binding domain specifically binding to CD3 as shown in SEQ ID NO.77 is deimmunized; see also WO2005/040220. Preferably, the arrangementof the V regions in this CD3-binding domain is VH-VL.

In another aspect, the invention relates to a bispecific single chainantibody comprising an amino acid sequence selected from the groupconsisting of:

-   -   (a) an amino acid sequence as depicted in any of SEQ ID NOs. 6,        8, 16, 18, 24, 26, 32, 34, 40, 42, 126, 130, 134 or 143;    -   (b) an amino acid sequence encoded by a nucleic acid sequence as        shown in SEQ ID NOs. 5, 7, 15, 17, 23, 25, 31, 33, 39, 41, 125,        129, 133 or 142;    -   (c) an amino acid sequence encoded by a nucleic acid sequence        hybridising under stringent conditions to the complementary        nucleic acid sequence of (b);    -   (d) an amino acid sequence encoded by a nucleic acid sequence        which is degenerate as a result of the genetic code to a        nucleotide sequence of (b); and    -   (e) an amino acid sequence at least 85% identical, more        preferred at least 90% identical, most preferred at least 95%        identical to the amino acid sequence of (a) or (b).

In one embodiment, the invention relates to a composition comprising abispecific single chain antibodies as defined above. Preferably, saidbispecific single chain antibodies as defined above are used aspharmaceutical compositions for the treatment of an epithelial tumor orepithelial tumors in a human. Said epithelial tumor(s) is (are)CEA-positive. The cytotoxic activity against CEA-positive epithelialtumor cells of these pharmaceutical compositions is resistant to evenhigh concentrations of soluble CEA antigen in the plasma of tumorpatients. Moreover, said bispecific single chain antibodies as definedabove or anti-CEA scFvs derived thereof may be used as diagnosticcompositions for the detection of an epithelial tumor or epithelialtumors in a human as set forth in more detail below.

The term “hybridizing under stringent conditions” as used herein refersto nucleic acid sequences capable of hybridizing, under stringenthybridization conditions, to sequences depicted in SEQ ID NOs. 5, 7, 15,17, 23, 25, 31, 33, 39, 41, 125, 129, 133 or 142, or the complementthereof, and which encode a bispecific single chain antibody havingcytotoxic activity against CEA-positive tumor cells. “Stringenthybridization conditions” refers to an overnight incubation at 42° C. ina solution comprising 50% formamide, 5×SSC (750 mM NaCl, 75 mM trisodiumcitrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10%dextran sulfate, and 20 μg/ml denatured, sheared salmon sperm DNA,followed by washing the filters in 0.1×SSC at about 65° C.

Whether any particular nucleic acid molecule or polypeptide is at least80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a nucleotide oramino acid sequence defined herein can be determined conventionallyusing known computer programs. A preferred method for determining thebest overall match between a query sequence (a sequence defined herein)and a subject sequence, also referred to as a global sequence alignment,can be determined using the FASTDB computer program based on thealgorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245(1990)). In asequence alignment the query and subject sequences are both DNAsequences. An RNA sequence can be compared by converting U's to T's.

The invention also provides for a pharmaceutical composition comprisinga nucleic acid sequence encoding a bispecific single chain antibody asdefined herein. Said nucleic acid can be utilized e.g. for gene therapyapproaches in order to treat an epithelial tumor in a human, as setforth in more detail below.

The invention further relates to a pharmaceutical composition comprisinga vector which comprises a nucleic acid sequence as defined above.Preferably, said vector further comprises a regulatory sequence which isoperably linked to said nucleic acid sequence defined above. Morepreferably, said vector is an expression vector.

Furthermore, the vector of the present invention may also be a genetransfer or gene targeting vector. Gene therapy, which is based onintroducing therapeutic genes or nucleic acids into cells by ex-vivo orin-vivo techniques is one of the most important applications of genetransfer. Suitable vectors, methods or gene-delivering systems forin-vitro or in-vivo gene therapy are described in the literature and areknown to the person skilled in the art; see, e.g., Giordano, NatureMedicine 2 (1996), 534-539; Schaper, Circ. Res. 79 (1996), 911-919;Anderson, Science 256 (1992), 808-813, Isner, Lancet 348 (1996),370-374; Muhlhauser, Circ. Res. 77 (1995), 1077-1086; Onodua, Blood 91(1998), 30-36; Verzeletti, Hum. Gene Ther. 9 (1998), 2243-2251; Verma,Nature 389 (1997), 239-242; Anderson, Nature 392 (Supp. 1998), 25-30;Wang, Gene Therapy 4 (1997), 393-400; Wang, Nature Medicine 2 (1996),714-716; WO 94/29469; WO 97/00957; U.S. Pat. No. 5,580,859; U.S. Pat.No. 5,589,466; U.S. Pat. No. 4,394,448 or Schaper, Current Opinion inBiotechnology 7 (1996), 635-640, and references cited therein. Thenucleic acid molecules and vectors as defined herein may be designed fordirect introduction or for introduction via liposomes, viral vectors(e.g. adenoviral, retroviral), electroporation, or other deliverysystems into the cell. Additionally, a baculoviral system can be used aseukaryotic expression system for the nucleic acid molecules as definedherein. The introduction and gene therapeutic approach should,preferably, lead to the expression of a functional bispecific singlechain antibody construct as defined herein, whereby said expressedbispecific single chain antibody construct is particularly useful in thetreatment, amelioration and/or prevention of an epithelial tumor in ahuman.

In a further aspect, the invention relates to a pharmaceuticalcomposition comprising a host transformed or transfected with a vectoror a nucleic acid as defined above.

A further aspect of the invention relates to a pharmaceuticalcomposition as defined hereinabove, further comprising a proteinaceouscompound capable of providing an activation signal for immune effectorcells.

Preferably, the pharmaceutical composition further comprises suitableformulations of carriers, stabilizers and/or excipients.

In another aspect, the invention relates to a process for the productionof a pharmaceutical composition as defined above, said processcomprising culturing a host as defined above under conditions allowingthe expression of the bispecific single chain antibody as definedhereinabove and recovering the produced bispecific single chain antibodyfrom the culture.

A further aspect of the invention relates to a use of a bispecificsingle chain antibody as defined hereinabove or as produced by theprocess as defined hereinabove, a nucleic acid molecule as definedhereinabove, a vector as defined hereinabove or a host as definedhereinabove for the preparation of a pharmaceutical composition for theprevention, treatment or amelioration of an epithelial tumor in a human.Another aspect of the invention relates to a method for the prevention,treatment or amelioration of an epithelial tumor in a human, said methodcomprising the step of administration of an effective amount of apharmaceutical composition of the invention or as produced according bythe process set forth above. The person skilled in the art, inparticular the attending physician can evaluate the successful treatmentof the patient in need of administration of the bispecificmolecule/bispecific single chain antibody of the invention. Accordingly,the administration scheme as well as the dosage and the administrationtime may be assessed by said person skilled in the art: A corresponding“amelioration” and/or “treatment” to be assessed is defined below.

As used herein, an “effective amount” or “therapeutically effectiveamount” of a pharmaceutical composition of the invention in the contextof epithelial tumors refers to that amount of the therapeutic agentsufficient to destroy, modify, control or remove primary, regional ormetastatic tumor tissue. A therapeutically effective amount may refer tothe amount of therapeutic agent sufficient to delay or minimize thespread of the epithelial tumor(s). A therapeutically effective amountmay also refer to the amount of the therapeutic agent or pharmaceuticalagent that provides a therapeutic benefit in the treatment or managementof the epithelial tumor(s). Further, a therapeutically effective amountwith respect to a therapeutic agent or pharmaceutical agent of theinvention means that amount of therapeutic agent or pharmaceutical agentalone, or in combination with other therapies, that provides atherapeutic benefit in the treatment or management of an epithelialtumor. Used in connection with an amount of the bispecific single chainantibody defined herein, the term can encompass an amount that improvesoverall therapy, reduces or avoids unwanted effects, or enhances thetherapeutic efficacy of or synergies (as defined herein) with anothertherapeutic agent. Preferably, a therapeutically effective amount of atherapeutic improves overall therapy, reduces or avoids unwantedeffects, or enhances the therapeutic efficacy of or synergies withanother therapeutic agent in the treatment of (an) epithelial tumor(s).For example, a bispecific single chain antibody as defined herein maycause a shrinkage of the diameter of an epithelial tumor of 20% ifadministered to a patient as a mono-therapy. In contrast, a secondtherapeutic e.g. an anti-cancer agent as defined below, may cause atumor shrinkage of 10%. However, if both the bispecific single chainantibody as defined herein and said second therapeutic are administeredin combination in form of a co-therapy, a tumor shrinkage of 50% may beobserved. Such an effect is understood as a synergestic effect as usedherein.

As referred to herein, the term “therapy” refers to any administrationscheme, method and/or agent that can be used in the prevention,treatment or amelioration of an epithelial tumor. The term “prevention,treatment or amelioration of an epithelial tumor” is set forth in moredetail below. The terms “therapies” and “therapy” may refer to abiological therapy, supportive therapy, chemotherapy, radiation therapyand/or other therapies useful in treatment, prevention, or ameliorationof an epithelial tumor, or one or more symptoms thereof.

As used herein, the terms “treat”, “treatment” and “treating” in thecontext of administering a therapy or therapies to a patient refer tothe reduction or amelioration of the progression, severity, and/orduration of an epithelial tumor. Said epithelial tumor(s) may beassociated with aberrant expression e.g., overexpression or activity ofCEA, and/or the amelioration of one or more symptoms thereof resultingfrom the administration of one or more therapies (including theadministration of one or more pharmaceutical or therapeutic agents).

The most preferred mode of administration is an intravenousadministration over a given time/time period. While the bispecificsingle chain antibody as defined herein may be administered per alone,preferred is administration in a pharmaceutically acceptable carrier.Examples of suitable pharmaceutical carriers are well known in the artand include phosphate buffered saline solutions, water, liposomes,various types of wetting agents, sterile solutions, etc. Compositionscomprising such carriers can be formulated by well known conventionalmethods. These pharmaceutical compositions can be administered to thesubject at a suitable dose. The dosage regimen will be determined by theattending physician and clinical factors. As is well known in themedical arts, dosages for any one patient depends upon many factors,including the patient's size, body surface area, age, the particularcompound to be administered, sex, time and route of administration,general health, and other drugs being administered concurrently.Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, and suspensions. Examples of non-aqueous solventsare propylene glycol, polyethylene glycol, and injectable organic esterssuch as ethyl oleate. Aqueous carriers include water, aqueous solutions,or suspensions, including saline and buffered media. Parenteral vehiclesinclude sodium chloride solution, Ringer's dextrose, dextrose and sodiumchloride, lactated Ringer's, or fixed oils. Intravenous vehicles includefluid and nutrient replenishes, electrolyte replenishers (such as thosebased on Ringer's dextrose), and the like. Preservatives and otheradditives may also be present such as, for example, antimicrobials,anti-oxidants, chelating agents, and inert gases and the like. Inaddition, the composition might comprise proteinaceous carriers, like,e.g., serum albumine or immunoglobuline, preferably of human origin. Itis envisaged that the co-therapy might comprise, in addition to theproteinaceous bispecific single chain antibody further biologicallyactive agents, depending on the intended use of the pharmaceuticalcomposition. Such agents might be agents acting on the gastrointestinalsystem, agents acting as cytostatica, agents preventing hyperurikemia,agents inhibiting immune reactions (e.g. corticosteroids, FK506), drugsacting on the circulatory system and/or agents such as T-cellco-stimulatory molecules or cytokines known in the art. Preferably, thebispecific single chain antibody as defined herein is formulated in abuffer, a stabilizer and a surfactant. The buffer may be a phosphate,citrate, succinate or acetate buffer. The stabilizer may be (an) aminoacid(s) and/or a sugar. The surfactants may be detergents, PEGs, or thelike. More preferably, the bispecific single chain antibody as definedherein is formulated in citrate, lysine, trehalose and Tween 80. As adiluent for said pharmaceutical composition, isotonic saline and Tween80 is preferred.

The term “amelioration” as used herein refers to an improvement or amoderation in the severity of a disease, i.e. an epithelial tumor. Forexample, such an amelioration may be the achievement of a stabledisease—or even more preferred—a shrinkage of the epithelial tumor(s),i.e. a minimal, partial response or complete response, due to theadministration of the pharmaceutical compositions of the invention.“Stable disease” refers to a disease state in which no or no significanttumor progression/growth can be observed or detected by clinical and/orhistological diagnostic methods. For example, a shrinkage of the tumorgreater than 50% shrinkage of the sum of cross-sectional areas of indexlesions may be considered as a “partial response”. A “complete response”denotes a state in which no lesion(s) can be detected any more aftertreatment. A response with a tumor shrinkage between stable disease andpartial response may be considered as a minimal response. For instance,a 20%, 25% or 30% shrinkage of the sum of cross-sectional areas of indexlesions may be referred to as a minimal response.

The term “amelioration” as used herein encompasses also a reduction ofthe number of epithelial tumors. It furthermore denotes theprevention/slowdown of tumor progression. Moreover, an improvement ofthe overall survival of treated tumor patients in comparison tonon-treated tumor patients may be considered as an “amelioration” asused herein. This applies mutatis mutandis to an improvement of theprogression-free survival or the relapse-free survival of treated tumorpatients as compared to non-treated tumor patients. In addition, theterm “amelioration” can also refer to a reduction of the intensity ofthe symptoms of an epithelial tumor, resulting e.g. in an improvement ofthe quality of life of the treated tumor patients.

The term “prevention of an epithelial tumor” as used herein is to beunderstood as follows: After surgical removal of the primary epithelialtumor(s) from a human patient and/or after chemotherapeutic orradiological treatment of the primary epithelial tumor(s), it may be thecase that not all tumor cells could be eliminated from the body.However, these remaining tumor cells may give rise to recurrent cancer,i.e. local recurrence and/or metastases in the patient. Metastasis is afrequent complication of cancer, yet the process through which cancercells disseminate from the primary tumor(s) to form distant colonies ispoorly understood. Metastatic cancers are almost without exceptionuncurable raising the necessity for new therapeutic modalities. Thepharmaceutical composition of the invention can be used to kill thesedisseminated tumor cells in order to prevent the formation of secondarytumors (originating from the tumor cells remaining in the body afterprimary therapy). In this way, the pharmaceutical composition helps toprevent the formation of local recurrence and/or metastases in tumorpatients.

The success of the anti-tumor therapy may be monitored by establishedstandard methods for the respective disease entities, e.g. bycomputer-aided tomography, X-ray, nuclear magnetic resonance tomography(e.g. for National Cancer Institute-criteria based response assessment[Cheson (1999), J. Clin. Oncol.; 17(4):1244]), positron-emissiontomography scanning, endoscopy, Fluorescence Activated Cell Sorting,aspiration of bone marrow, pleural or peritoneal fluid,tissue/histologies, and various epithelial tumor specific clinicalchemistry parameters (e.g. soluble CEA concentration in serum) and otherestablished standard methods may be used. In addition, assaysdetermining T cell activation may be used; see e.g. WO99/054440.Statistics for the determination of overall survival, progression-freesurvival or relapse-free survival of treated tumor patients incomparison to non-treated tumor patients may also be used.

Preferably, said epithelial tumor is a gastrointestinal adenocarcinoma,a breast adenocarcinoma or a lung adenocarcinoma. Said gastrointestinaladenocarcinoma is more preferably a colorectal, pancreatic, anoesophageal or a gastric adenocarcinoma.

Even more preferred, said pharmaceutical composition of the invention isfor the treatment of progressive tumors, late stage tumors, tumorpatients with high tumor load/burden, metastatic tumors, or tumorpatients with a CEA serum concentration higher than 100 ng/ml (asdetermined e.g. by ELISA).

In another preferred embodiment of the uses or methods of the invention,said pharmaceutical composition as defined hereinabove is suitable to beadministered in combination with an additional drug, i.e. as part of aco-therapy.

In certain embodiments, the bispecific single chain antibody orpharmaceutical composition as defined herein is administered incombination with one or more other therapies. In certain embodiments,the bispecific single chain antibody or pharmaceutical composition asdefined herein is administered to a patient concurrently with one ormore other therapies. Preferably, such therapies are useful for thetreatment of epithelial tumors. The term “concurrently” is not limitedto the administration of pharmaceutical compositions or therapeuticagents at exactly the same time, but rather it is meant that thebispecific single chain antibody or pharmaceutical composition asdefined herein and the other agent(s) are administered to a patient in asequence and within a time interval such that the bispecific singlechain antibody or pharmaceutical composition as defined herein can acttogether with the other agent to provide an increased benefit than ifthey were administered otherwise. For example, each therapeutic agentmay be administered at the same time or sequentially in any order atdifferent points in time; however, if not administered at the same time,they should be administered sufficiently close in time so as to providethe desired therapeutic effect.

Each therapeutic agent can be administered separately, in anyappropriate form and by any suitable route. In other embodiments, thebispecific single chain antibody or pharmaceutical composition asdefined herein are administered before, concurrently or after surgery.Preferably the surgery completely removes localized epithelial tumors orreduces the size of large epithelial tumors. Surgery can also be done asa preventive measure or to relieve pain.

The dosage amounts and frequencies of administration provided herein areencompassed by the term “therapeutically effective” as defined above.The dosage and frequency further will typically vary according tofactors specific for each patient depending on the specific therapeuticor prophylactic agents administered, the severity and type of epithelialtumor, the route of administration, as well as age, body weight,response, and the past medical history of the patient. Suitable regimenscan be selected by one skilled in the art by considering such factorsand by following, for example, dosages reported in the literature andrecommended in the Physicians' Desk Reference (59th ed., 2005).

In some embodiments, therapy by administration of the bispecific singlechain antibody or pharmaceutical composition as defined herein iscombined with the administration of one or more therapies such aschemotherapies, radiation therapies, hormonal therapies, and/orbiological therapies/immunotherapies. Therapeutic agents include, butare not limited to, proteinaceous molecules, including, but not limitedto, peptides, polypeptides, proteins, including post-translationallymodified proteins, antibodies etc.; or small molecules (less than 1000daltons), inorganic or organic compounds; or nucleic acid moleculesincluding double-stranded or single-stranded DNA, or double-stranded orsingle-stranded RNA, as well as triple helix nucleic acid molecules.Therapeutic agents can be derived from any known organism (including,but not limited to, animals, plants, bacteria, fungi, and protista, orviruses) or from a library of synthetic molecules.

In a specific embodiment, the methods and uses of the inventionencompass administration of the bispecific single chain antibody orpharmaceutical composition as defined herein in combination with theadministration of one or more therapeutic agents that are inhibitors ofkinases such as Gefitinib (Iressa), Erlotinib (Tarceva),anti-EGFR-antibodies (e.g. Cetuximab; Erbitux), oranti-Her2/neu-antibodies (e.g. Trastuzumab; Herceptin) described in theart; see e.g., Hardie and Hanks (1995) The Protein Kinase Facts Book, Iand II, Academic Press, San Diego, Calif.

In another specific embodiment, the methods and uses of the inventionencompass administration of the bispecific single chain antibody orpharmaceutical composition as defined herein in combination with theadministration of one or more therapeutic agents that are angiogenesisinhibitors such as anti-VEGF-antibodies (e.g. Bevacizumab; Avastin),small molecular compounds (e.g. Vatalanib or Sorafenib) orCOX-inhibitors described in the art.

In another specific embodiment, the methods and uses of the inventionencompass administration of the bispecific single chain antibody orpharmaceutical composition as defined herein in combination with theadministration of one or more therapeutic agents that are anti-canceragents such as 5-Fluorouracil, Leucovorin, Capecitabine, Oxaliplatin,Irinotecan, Gemcitabine, Doxorubicin, Epirubicin, Etoposide, Cisplatin,Carboplatin, Taxanes (e.g. Docetaxel, Paclitaxel) described in the art.

Preferably, a co-therapy of a patient with an epithelial tumor using abispecific single chain antibody or pharmaceutical composition asdefined herein in combination with (a) further therapeutic agent(s)results in an synergistic effect. As used herein, the term “synergistic”refers to a combination of therapies (e.g., a combination of abispecific single chain antibody as defined herein and (a) furthertherapeutic agent(s) as set forth above) which is more effective thanthe additive effects of any two or more single therapies (e.g., one ormore therapeutic agents). For example, a bispecific single chainantibody as defined herein may cause a shrinkage of the diameter of anepithelial tumor of 20% if administered to a patient as a mono-therapy.In contrast, a second therapeutic e.g. an anti-cancer agent as definedbelow, may cause a tumor shrinkage of 10%. However, if both thebispecific single chain antibody as defined herein and said secondtherapeutic are administered in combination in form of a co-therapy, atumor shrinkage of 50% may be observed.

A synergistic effect of a combination of therapies (e.g., a combinationof a bispecific single chain antibody as defined herein and (a) furthertherapeutic agent(s) as set forth above) permits the use of lowerdosages of one or more of therapies (e.g., one or more therapeuticagents) and/or less frequent administration of said therapies to apatient with a disease, e.g. an epithelial tumor. The ability to utilizelower dosages of therapies (e.g., therapeutic agents) and/or toadminister said therapies less frequently reduces the toxicityassociated with the administration of said therapies to a subjectwithout reducing the efficacy of said therapies in the prevention ortreatment of a disease, e.g. an epithelial tumor. In addition, asynergistic effect can result in improved efficacy of therapies (e.g.,therapeutic agents) in the prevention, management, treatment and/oramelioration of an epithelial tumor (which may be associated withaberrant expression (e.g., overexpression) or activity of CEA). Finally,synergistic effect of a combination of therapies (e.g., therapeuticagents) may avoid or reduce adverse or unwanted side effects associatedwith the use of any single therapy.

In said co-therapy, an active agent may be optionally included in thesame pharmaceutical composition as the bispecific single chain antibodydefined herein, or may be included in a separate pharmaceuticalcomposition. In this latter case, said separate pharmaceuticalcomposition is suitable for administration prior to, simultaneously asor following administration of said pharmaceutical compositioncomprising the bispecific single chain antibody as defined herein. Theadditional drug or pharmaceutical composition may be a non-proteinaceouscompound or a proteinaceous compound. In the case that the additionaldrug is a proteinaceous compound, it is advantageous that theproteinaceous compound be capable of providing an activation signal forimmune effector cells.

Preferably, said proteinaceous compound or non-proteinaceous compoundmay be administered simultaneously or non-simultaneously with abispecific single chain antibody as defined hereinabove, a nucleic acidmolecule as defined hereinabove, a vector as defined as definedhereinabove, or a host as defined as defined hereinabove. Preferably,said subject to be treated is a human.

In a further embodiment, a single chain bispecific antibody or anti-CEAscFvs as defined herein may be conjugated to a diagnostic or detectableagent. Such diagnosis and detection can be accomplished by coupling theantibody or scFv to detectable substances for example to variousenzymes, such as horseradish peroxidase, alkaline phosphatase,beta-galactosidase, or acetylcholinesterase; prosthetic groups, such asstreptavidin/biotin and avidin/biotin; fluorescent materials, such as,umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin;luminescent materials, such as, luminol; bioluminescent materials, suchas, luciferase, luciferin, and aequorin; radioactive materials andisotopes, such as cobalt (57Co), indium (115In, 113In, 112In, 111In),iodine (131I, 125I, 123I, 121I), or yttrium (90Y), positron emittingmetals using various positron emission tomographies, and nonradioactiveparamagnetic metal ions.

Techniques for conjugating moieties to antibodies are well known.Moieties can be conjugated to antibodies by any method known in the art,including, but not limited to aldehyde/Schiff linkage, sulphydryllinkage, acid-labile linkage, cis-aconityl linkage, hydrazone linkage,enzymatically degradable linkage; see generally Garnett, 2002, Adv. DrugDeliv. Rev. 53:171-216. Additional techniques for conjugating moietiesto antibodies are well known, see, e.g., Arnon et al., MonoclonalAntibodies For Immunotargeting Of Drugs In Cancer Therapy. In MonoclonalAntibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (AlanR. Liss, Inc. 1985). Methods for fusing or conjugating antibodies topolypeptide moieties are known in the art; see, e.g.; Ashkenazi et al.,1991, PNAS 88: 10535-10539. The fusion of an antibody to a moiety doesnot necessarily need to be direct, but may occur through linkersequences. Such linker molecules are commonly known in the art anddescribed in Denardo et al., 1998, Clin Cancer Res. 4:2483-90; Petersonet al., 1999, Bioconjug. Chem. 10:553.

In a further aspect, the invention relates to a kit comprising abispecific single chain antibody as defined hereinabove, a nucleic acidmolecule as defined hereinabove, a vector as defined hereinabove, or ahost as defined hereinabove.

These and other embodiments are disclosed and encompassed by thedescription and Examples of the present invention. Recombinanttechniques and methods in immunology are described e.g. in Sambrook etal. Molecular Cloning: A Laboratory Manual; Cold Spring HarborLaboratory Press, 3^(rd) edition 2001; Lefkovits; Immunology MethodsManual; The Comprehensive Sourcebook of Techniques; Academic Press,1997; Golemis; Protein-Protein Interactions: A Molecular Cloning Manual;Cold Spring Laboratory Press, 2002. Further literature concerning anyone of the antibodies, methods, uses and compounds to be employed inaccordance with the present invention may be retrieved from publiclibraries and databases, using for example electronic devices. Forexample, the public database “Medline”, available on the Internet, maybe utilized, for example under http://www.ncbi.nlm.nih.qov/PubMed/medline.html. Further databases and addresses, such ashttp://www.ncbi.nim.nih.qov/, http://www.infobioaen.fr/,http://www.fmi.ch/bioloqv/researchtools.html, http://www.tiqr.orQ/. areknown to the person skilled in the art and can also be obtained using,e. g., http://www.lvcos.com. For tumor-related topics see e.g.http://www.nih.gov or http://www.dkfz.de.

The Figures show:

FIG. 1: FACS binding analysis of various human CEA-reactive bispecificsingle chain constructs to CHO cells transfected with human CEA and CD3positive HPB-All cells, respectively. As a positive control for bindingto CEA, monoclonal antibody Col-1 has been used. For control of bindingto human CD3, a CD19×CD3 bispecific single chain construct as describedin WO 99/054440 was used. In this positive control, the thick linerepresents cells incubated with 10 μg/ml purified CD19×CD3 bispecificsingle chain antibody that was subsequently incubated with the anti-Hisantibody and the detection antibody. The thin histogram line reflectsthe negative control: cells incubated with the anti-His antibody and thedetection antibody. Binding activity for human (membrane-bound) CEA andhuman CD3 were detectable for CEAI VHVL×SEQ ID NO.77 VHVL, CEAI VLVH×SEQID NO.77 VHVL, CEAII VHVL×SEQ ID NO.77 VHVL, CEAIII VLVH×SEQ ID NO.77VHVL and CEAIII VHVL×SEQ ID NO.77 VHVL. In the respective histogramscorresponding to the bispecific single chain antibodies as described inthe invention, the thin line represents the negative control, the brightthick line represents cells incubated with culture supernatant, whereasthe dark thick (most right) line represents cells incubated with 10μg/ml purified bispecific single chain antibody.

FIG. 2: Binding signals of bispecific single chain anti-CEA/anti-CD3antibodies CEAI VHVL×SEQ ID NO. 77 VHVL, CEAII VHVL×SEQ ID NO. 77 VHVLand CEAIII VHVL×SEQ ID NO. 77 VHVL and anti-CEA antibody Col-1 tosoluble CEA detected by direct ELISA. CEAI VHVL×SEQ ID NO. 77 VHVL(anti-CEA binding domain derived from mAb A5B7), CEAII VHVL×SEQ ID NO.77VHVL (anti-CEA binding domain derived from mAb T84.66), and CEAIIIVHVL×SEQ ID NO.77 VHVL (anti-CEA binding domain derived from mAb MFE-23)bispecific single chain antibodies and the mouse monoclonal antibodyCol-1 specifically bound to immobilized soluble human CEA. No bindingsignal was observed in the absence of the soluble CEA antigen (PBScontrol).

FIG. 3: The indicated CEA-reactive bispecific single chain constructsredirected T cells to lyse CHO cells transfected with CEA, in theabsence of soluble CEA. Stimulated human CD8 positive CTLs were used aseffector cells. To demonstrate the specificity of the redirected lysis,a non-CEA reactive bispecific single chain construct was included asnegative control. Cytotoxic activity against human CEA-transfectedtarget cells (CHO-CEA⁺ cells) for various domain arrangements, i.e. forSEQ ID NO.77 VHVL×CEAI VHVL and SEQ ID NO.77 VHVL×CEAI VLVH (bothconstructs with anti-CD3 binding domain N-terminally), as well as forCEAI VLVH×SEQ ID NO.77 VHVL and CEAI VHVL×SEQ ID NO.77 VHVL (anti-CD3binding domain C-terminally) could be shown. Non-transfected CHO cells(lacking human CEA) were used as a negative control.

FIG. 4: The indicated CEA-reactive bispecific single chain constructsredirected T cells to lyse CHO cells transfected with CEA, in theabsence of soluble CEA. To demonstrate the specificity of the redirectedlysis, non-transfected CHO cells were included as negative control.Stimulated human CD8 positive CTLs were used as effector cells. CEA I-HL(CEAI VHVL×SEQ ID NO.77 VHVL), CEA III-LH (CEAIII VLVH×SEQ ID NO.77VHVL), CEA III-HL (CEAIII VHVL×SEQ ID NO.77 VHVL), and CEA II HL (CEAIIVHVL×SEQ ID NO.77 VHVL) showed cytotoxic activity against humanCEA-transfected CHO cells. Non-transfected CHO cells (lacking human CEA)were used as a negative control for CEA I-LH (CEAI VLVH×SEQ ID NO.77VHVL), CEA III-HL (CEAIII VHVL×SEQ ID NO.77 VHVL) and CEA II HL (CEAIIVHVL×SEQ ID NO.77 VHVL). CEAI denotes a variable region derived frommurine mAb A5B7, CEAII is a variable region derived from murine mAbT84.66 and CEAIII refers to a variable region from murine mAb MFE-23.

FIG. 5: The indicated CEA-reactive bispecific single chain constructsredirected T cells to lyse CHO cells transfected with CEA in thepresence of soluble human CEA. Stimulated human CD8 positive CTLs wereused as effector cells. The cytotoxic activity mediated by CEAI VLVH×SEQID NO.77 VHVL and CEAI VHVL×SEQ ID NO.77 VHVL is not inhibited byincreasing amounts of soluble human CEA, up to 1 μg/ml. CEAI is avariable region derived from murine mAb A5B7.

FIG. 6: The indicated CEA-reactive bispecific single chain constructsredirected T cells to lyse CHO cells transfected with CEA in thepresence of soluble human CEA. Stimulated human CD8 positive CTLs wereused as effector cells. The cytotoxic activity mediated by SEQ ID NO.77VHVL×CEAI VHVL and SEQ ID NO.77 VHVL×CEAI VLVH is not inhibited byincreasing amounts of soluble human CEA, up to 1 μg/ml. CEAI is avariable region derived from murine mAb A5B7.

FIG. 7: The indicated CEA-reactive bispecific single chain constructredirected T cells to lyse CHO cells transfected with CEA in thepresence of soluble human CEA. Stimulated human CD8 positive CTLs wereused as effector cells. Cytotoxic activity of CEAII VHVL×SEQ ID NO.77VHVL is inhibited by increasing amounts of soluble CEA. CEAII VHVL isderived from mAb T84.66.

FIG. 8: The indicated CEA-reactive bispecific single chain constructsredirected T cells to lyse CHO cells transfected with CEA in thepresence of soluble human CEA. Stimulated human CD8 positive CTLs wereused as effector cells. Whereas CEAI VHVL×SEQ ID NO.77 VHVL-mediatedcytotoxicity is resistant to inhibition by soluble CEA antigen, CEAIIIVHVL×SEQ ID NO.77 VHVL-mediated cytotoxic activity is inhibited by evenlow amounts of soluble CEA. CEAIII VHVL is derived from mAb MFE-23,whereas CEAI is a variable region derived from murine mAb A5B7.

FIG. 9: The indicated CEA-reactive bispecific single chain constructsredirected T cells to lyse Kato III cells in the presence of increasingamounts of soluble CEA antigen. Native human PBMCs were used as effectorcells. CEAII VHVL×SEQ ID NO. 77-mediated cytotoxic activity is notresistant to soluble CEA. CEAII VHVL is derived from mAb T84.66.

FIG. 10: The indicated CEA-reactive bispecific single chain constructsredirected T cells to lyse Kato III cells in the presence of increasingamounts of soluble CEA antigen. Stimulated human CD8 positive CTLs wereused as effector cells. CEAI VHVL×SEQ ID NO.77 VHVL-mediatedcytotoxicity is resistant to soluble CEA. In contrast, CEAII VHVL×SEQ IDNO.77 VHVL-mediated cytotoxic activity is inhibited by increasingamounts of soluble CEA. CEAII VHVL is derived from mAb T84.66, whereasCEAI is a variable region derived from murine mAb A5B7.

FIG. 11: The indicated CEA-reactive bispecific single chain constructsredirected T cells to lyse CHO cells transfected with CEA in thepresence of increasing amounts of soluble CEA antigen. Stimulated humanCD8 positive CTLs were used as effector cells. CEAII VHVL×SEQ ID NO.77VHVL-mediated cytotoxic activity is inhibited by increasing amounts ofsoluble CEA. CEAII VHVL is derived from mAb T84.66.

FIG. 12: Flow cytometric analysis of periplasmic preparations containingFlag-tagged scFv protein fragments from selected clones. Periplasmicpreparations of soluble scFv protein fragments were added to 100,000 to200,000 CEA-transfected CHO cells. For detection a monoclonal anti-Flagantibody was used followed by a PE-labeled polyclonal anti-mouseantibody. ScFvs binding to cells was measured by an increase influorescence intensity as compared to cells that were incubated with PBSalone. Fluorescence intensity is blotted on the X-axis, the number ofevents is blotted on the Y-axis. The negative control (PBS and detectionreagents) is shown as filled curve, the respective scFvs are shown asgrey lines. Shifting to the right indicates positive binding to thecells. All of the scFvs, i.e. A-121, A-183, A-240, A-313, A-290, A-315,A4-35, A4-52 and MP2-A5, bind to membrane-bound CEA on CHO cells. Eachof the scFv consists of the murine A5B7 VH region and a human VL region,as described in Example 6.

FIG. 13: Flow cytometric analysis of periplasmic preparations containingFlag-tagged scFv protein fragments from selected. Periplasmicpreparations of soluble scFv protein fragments were added to 100,000 to200,000 CEA-transfected CHO cells. Detection was performed by amonoclonal anti-Flag antibody followed by a PE-labeled polyclonalanti-mouse antibody. ScFvs binding to cells was measured by an increasein fluorescence intensity as compared to cells that were incubated withPBS alone. Fluorescence intensity is blotted on the X-axis, the numberof events is blotted on the Y-axis. The negative control (PBS anddetection reagents) is shown as filled curve, the respective scFvs areshown as grey lines. Shifting to the right indicates positive binding tothe cells. The fully human scFv constructs MP510_3-A5.3, MP510_3-B9.1,and MP510_3-D8.1 bind to membrane-bound CEA on CHO cells. Each of thesescFvs consists of a human VH region and the human VL region A240, asdescribed in Example 7. 240 Vlambda.3 is a scFv consisting of the murineA5B7 VH region and the human VL A-240 region. This construct shows alsoCEA-binding activity.

FIG. 14: FACS binding analysis of various human CEA-reactive bispecificsingle chain constructs to Kato III cells and HPB-All cells,respectively. The thick line represents cells incubated with cellculture supernatant of transfected CHO cells incubated with the anti-Hisantibody and the detection antibody. The thin histogram line reflectsthe negative control: cells incubated with the anti-His antibody and thedetection antibody. The human bispecific single chain antibodyconstructs A5 VH-A240 VL×SEQ ID NO.77 VHVL, B9 VH-A240 VL×SEQ ID NO.77VHVL, and D8 VH-A240 VL×SEQ ID NO.77 VHVL bind to human CEA on Katocells and to human CD3 on HPB-All cells. CEAI VH-A240 VL×SEQ ID NO.77VHVL with the VH region of the CEA binding domain derived from mAb A5B7shows the same binding activity.

FIG. 15: Cytotoxicity assay of the indicated CEA-reactive bispecificsingle chain constructs redirected to CHO cells transfected with CEA, inthe absence of soluble CEA. Stimulated human CD8 positive CTLs were usedas effector cells. Cytotoxic activity could be detected for A5 VH-A240VL×SEQ ID NO.77 VHVL, B9 VH-A240 VL×SEQ ID NO.77 VHVL, D8 VH-A240 VL×SEQID NO.77 VHVL and CEAI VH-A240 VL×SEQ ID N0.77 VHVL. CEAI VH is a VHregion derived from mAb A5B7.

FIG. 16: Cytotoxicity assay of the indicated CEA-reactive bispecificsingle chain constructs redirected to CHO cells transfected with CEA, inthe absence of soluble CEA. Stimulated human CD8 positive CTLs were usedas effector cells. This Figure demonstrates cytotoxic activity for A240VL-A5 VH×SEQ ID NO.77 VHVL, A240 VL-B9 VH×SEQ ID NO.77 VHVL, A240 VL-D8VH×SEQ ID NO.77 VHVL, and A240 VL-CEAI VH×SEQ ID NO.77 VHVL.

FIG. 17: Cytotoxicity assay of the indicated CEA-reactive bispecificsingle chain constructs redirected to CHO cells transfected with CEA, inthe absence of soluble CEA. Stimulated human CD8 positive CTLs were usedas effector cells. Cytotoxicity against CEA+ target cells is shown forSEQ ID NO.77 VHVL×A5 VH-A240 VL, SEQ ID NO.77 VHVL×B9 VH-A240 VL, SEQ IDNO.77 VHVL×D8 VH-A240 VL, SEQ ID NO.77 VHVL×CEAI VH-A240 VL and SEQ IDNO.77 VHVL×CEAI VLVH.

FIG. 18: Cytotoxicity assay of the indicated CEA-reactive bispecificsingle chain constructs redirected to CHO cells transfected with CEA, inthe absence of soluble CEA. Stimulated human CD8 positive CTLs were usedas effector cells. Cytotoxic activity is shown for SEQ ID NO.77VHVL×A240 VL-A5 VH, SEQ ID NO.77 VHVL×A240 VL-B9 VH, SEQ ID NO.77VHVL×A240 VL-D8 VH, and SEQ ID NO.77 VHVL×A240VL-CEAI VH and SEQ IDNO.77 VHVL×CEAI VLVH.

FIG. 19: Cytotoxicity assay of the indicated CEA-reactive bispecificsingle chain constructs redirected to CHO cells transfected with CEA inthe presence of increasing amounts of soluble CEA antigen. Stimulatedhuman CD8 positive CTLs were used as effector cells. The Figuredemonstrates the resistance of cytotoxic activity of human bispecificsingle chain antibody constructs to soluble CEA antigen, as exemplifiedfor A5 VH-A240 VL×SEQ ID NO.77 VHVL and B9 VH-A240 VL×SEQ ID NO.77 VHVL.

FIG. 20: Cytotoxicity assay of the indicated CEA-reactive bispecificsingle chain constructs redirected to CHO cells transfected with CEA inthe presence of increasing amounts of soluble CEA antigen. Stimulatedhuman CD8 positive CTLs were used as effector cells. Human bispecificsingle chain antibody constructs D8 VH-A240 VL×SEQ ID NO.77 VHVL andCEAI VH-A240 VL×SEQ ID NO.77 VHVL also show resistance to soluble CEAantigen.

FIG. 21: The indicated CEA-reactive bispecific single chain constructsredirected T cells to lyse CHO cells transfected with CEA, in theabsence of soluble CEA. To demonstrate the specificity of the redirectedlysis, non-transfected CHO cells were included as negative control.Stimulated human CD8 positive CTLs were used as effector cells. A240VL-B9 VH×SEQ ID NO.77 VHVL, SEQ ID NO.77 VHVL×A240 VL-B9 VH, SEQ IDNO.77 VHVL×B9 VH-A240 VL, B9 VH-A240 VL×SEQ ID NO.77 VHVL, and SEQ IDNO.77 VHVL×CEA I VHVL revealed cytotoxic activity against humanCEA-transfected CHO cells.

FIG. 22: The indicated CEA-reactive bispecific single chain constructredirected T cells to lyse CHO-CEA+ cells in the presence of increasingamounts of soluble CEA antigen. Stimulated human CD8+ T cells were usedas effector cells. A240 VL-B9 VH×SEQ ID NO.77 VHVL-mediated cytotoxicactivity is resistant to soluble CEA.

FIG. 23: High resolution cation exchange chromatogram of the bispecificsingle chain construct A240 VL-B9 VH×SEQ ID NO.77 VHVL, the blue line(upper curve) shows the overall charge isoforms of the protein. Onesingle peak was detected showing high homogeneity of the construct.

FIG. 24: Protein stability assay based on the assessment of cytotoxicityafter incubation in human plasma for 24 h. The CEA-reactive bispecificsingle chain construct redirected to CHO cells transfected with CEA, inthe absence of soluble CEA. Stimulated human CD8 positive CTLs were usedas effector cells. The Figure demonstrates the plasma stability of thebispecific single chain construct A240 VL-B9 VH×SEQ ID NO.77 VHVL inhuman plasma. Cytotoxic activity of the construct is not influenced byplasma proteins under physiological conditions.

FIG. 25: High resolution cation exchange chromatogram of the bispecificsingle chain construct SEQ ID NO.77 VHVL×E12 VH-A240 VL, the blue line(upper curve) shows the overall charge isoforms of the protein. TheFigure demonstrates the homogeneity of the bispecific single chainconstruct SEQ ID NO.77 VHVL×E12 VH-A240 VL.

FIG. 26: Protein stability assay based on the assessment of cytotoxicityafter incubation in human plasma for 24 h. The CEA-reactive bispecificsingle chain construct redirected to CHO cells transfected with CEA, inthe absence of soluble CEA. Stimulated human CD8 positive CTLs were usedas effector cells. This Figure demonstrates the plasma stability of thebispecific single chain construct SEQ ID NO.77 VHVL×E12 VH-A240 VL inhuman plasma. Cytotoxic activity of the construct is not influenced byplasma proteins under physiological conditions.

FIG. 27: The indicated CEA-reactive bispecific single chain constructredirected T cells to lyse CHO-CEA+ cells in the presence of increasingamounts of soluble CEA antigen. Stimulated human CD8+ T cells were usedas effector cells. The bispecific single chain construct SEQ ID NO.77VHVL×E12 VH-A240 VL mediated cytotoxic activity that is resistant tosoluble CEA.

The following Examples illustrate the invention:

EXAMPLE 1: GENERATION OF CHO CELLS TRANSFECTED WITH HUMAN CEA(CARCINOEMBRYONIC ANTIGEN-RELATED CELL ADHESION MOLECULE 5; CEACAM5)

CEA-positive Kato III cells (human gastric carcinoma cell line; ATCCHTB-103) were used to obtain the total RNA that was isolated accordingto the instructions of the kit manual (Qiagen, RNeasy Mini Kit). Theobtained RNA was used for cDNA synthesis by random-primed reversetranscription. For cloning of the full length sequence of the CEAantigen, the following oligonucleotides were used: 5′ CEACAM5 EcoRIGAATTCGCCACCATGGAGTCTCCCTCGGCCCC (SEQ ID NO. 74) and 3′ CEACAM5 Sal IGTCGACCTATATCAGAGCAACCCC (SEQ ID NO. 75). A PCR (denaturation at 93° C.for 5 min, annealing at 58° C. for 1 min, elongation at 72° C. for 1 minfor the first cycle; denaturation at 93° C. for 1 min, annealing at 58°C. for 1 min, elongation at 72° C. for 1 min for 30 cycles; terminalextension at 72° C. for 5 min) was used to amplify the coding sequence.The PCR product was subsequently digested with EcoRI and SalI, ligatedinto the appropriately digested expression vector pEFDHFR, andtransformed into E. coli. The isolated plasmid DNA was sequenced andcompared with the established nucleotide sequence of CEACAM5 (NM_004363at the National Center for biotechnology information,http://www.ncbi.nlm.nih.gov/) The aforementioned procedures were carriedout according to standard protocols (Sambrook, Molecular Cloning; ALaboratory Manual, Cold Spring Harbour Laboratory Press, Cold SpringHarbour, N.Y. (1989; 2001). The clone with the verified nucleotidesequence was transfected into DHFR deficient CHO cells for eukaryoticexpression of the construct. Eukaryotic protein expression in DHFRdeficient CHO cells was performed as described in Kaufmann (Kaufmann R.J., Methods Enzymol. 185 (1990), 537-566). Gene amplification of theconstruct was induced by increasing concentrations of MTX to a finalconcentration of up to 20 nM MTX. The transfected cells were then testedfor expression of CEA antigen using an FACS assay. For that purpose,2.5×10⁵ transfected cells were incubated with 5 μg/ml of the murinemonoclonal antibody COL-1 (No. MS-613-P1ABX, Neomakers; Fremont, Calif.,USA). The binding of the antibody was detected with aR-Phycoerythrin-conjugated affinity purified F(ab′)2 fragment, goatanti-mouse IgG, Fc-gamma fragment specific antibody, diluted 1:100 in 50μl PBS with 2% FCS (obtained from Dianova, Hamburg, Germany). Cells wereanalyzed by flow cytometry on a FACS-Calibur (Becton Dickinson,Heidelberg). FACS staining and measuring of the fluorescence intensitywere performed as described in Current Protocols in Immunology (Coligan,Kruisbeek, Margulies, Shevach and Strober, Wiley-Interscience, 2002). Asa result, the transfectants demonstrated a clearly positive staining forthe human CEA antigen.

EXAMPLE 2: GENERATION OF CEA×CD3 BISPECIFIC SINGLE CHAIN ANTIBODIES

Generally, bispecific single chain antibody molecules, each comprising adomain with binding specificity for the human CEA antigen as well as adeimmunized domain with binding specificity for the human CD3 antigendepicted in SEQ ID NO.77 were designed as set out in Table 1. Thearrangement of the V regions in this CD3 binding domain is always VH-VL.This de-immunised anti-CD3 binding domain used in the bispecific singlechain antibodies as defined herein has been described previously, e.g.in WO2005/040220.

1. Formats of Bispecific Single Chain Antibody Molecules ComprisingAnti-CEA and Anti-CD3 Specificities (Table 1)

SEQ ID NO. (amino acid Formats of protein constructs sequence) (Nterminus→ C terminus) 2 SEQ ID NO. 77 VHVL × CEA I VLVH 4 SEQ ID NO. 77VHVL × CEA I VHVL 6 CEA I VLVH × SEQ ID NO. 77 VHVL 8 CEA I VHVL × SEQID NO. 77 VHVL 10 CEA II VHVL × SEQ ID NO. 77 VHVL 12 CEA III VLVH × SEQID NO. 77 VHVL 14 CEA III VHVL × SEQ ID NO. 77 VHVL 16 CEA I VH-A240VL ×SEQ ID NO. 77 VHVL 18 A240VL − CEA I VH × SEQ ID NO. 77 VHVL 20 SEQ IDNO. 77 VHVL × CEA I VH − A240 VL 22 SEQ ID NO. 77 VHVL × A240 VL − CEA IVH 24 A5 VH − A240 VL × SEQ ID NO. 77 VHVL 26 A240 VL − A5 VH × SEQ IDNO. 77 VHVL 28 SEQ ID NO. 77 VHVL × A240 VL − A5 VH 30 SEQ ID NO. 77VHVL × A5 VH − A240 VL 32 B9 VH − A240 VL × SEQ ID NO. 77 VHVL 34 A240VL − B9 VH × SEQ ID NO. 77 VHVL 36 SEQ ID NO. 77 VHVL × B9 VH − A240 VL38 SEQ ID NO. 77 VHVL × A240 VL − B9 VH 40 D8 VH − A240 VL × SEQ ID NO.77 VHVL 42 A240 VL − D8 VH × SEQ ID NO. 77 VHVL 44 SEQ ID NO. 77 VHVL ×D8 VH − A240 VL 46 SEQ ID NO. 77 VHVL × A240 VL − D8 VH 126 A5 VH-A240VL# × SEQ ID NO. 77 VHVL 128 SEQ ID NO. 77 VHVL × A5 VH-A240VL# 130 B9VH-A240 VL# × SEQ ID NO. 77 VHVL 132 SEQ ID NO. 77 VHVL × B9 VH-A240VL#134 D8 VH-A240 VL# × SEQ ID NO. 77 VHVL 136 SEQ ID NO. 77 VHVL × D8VH-A240VL# 143 SEQ ID NO. 77 VHVL × E12 VH-A240VL

The aforementioned constructs containing the variable light-chain (VL)and variable heavy-chain (VH) regions specific for the human CEA antigenderived from monoclonal antibodies, hybridomas or obtained by phagedisplay guided selection (PDGS) were obtained by gene synthesis andsubsequent cloning into an expression vector comprising the CD3-specificVH and VL combinations. The generation of said bispecific single chainconstructs can also be carried out according to recombinant techniquesdescribed e.g. in Sambrook (loc.cit.). A detailed instruction thereforis provided for e.g. in WO 99/054440.

The anti-CD3 binding domain corresponds to a de-immunized domain withbinding specificity for the human CD3 antigen. The arrangement of the Vregions of the deimmunized anti-CD3 binding domain in the bispecificsingle chain constructs described herein is always VH-VL. Thecorresponding amino acid sequence of said VH-VL domain is depicted inSEQ ID NO. 77. CEAI, CEAII and CEAIII specific for the humancarcinoembryonic antigen contain the variable light-chain (VL) andvariable heavy-chain (VH) regions derived from mAbs A5B7 (Chester, K. A.et al., Int J Cancer 57 (1994), 67-72), T84.66 (Neumaier, M. et al.,Cancer Res 50 (1990), 2128-34) and MFE-23 (Boehm, M. K. Biochem J 2(2000), 519-28), respectively. A5, B9, D8, and E12 are human VH regionsspecific for human CEA, whereas A240 is a human VL region with the samespecificity. The generation of the human A5, B9, D8, E12 and A240 Vregions is described in detail in Examples 6 and 7. The correspondingnucleotide and amino acid sequences of all bispecific single chainantibodies described herein are shown in the sequence listing.

In the following the generation of the CEA I VLVH×SEQ ID NO.77 VHVL (SEQID NO.6) construct is described in more detail. The generation of theother constructs mentioned above can be performed accordingly with thenecessary implementation of modifications to the methods being well inthe scope of the person skilled in the art.

To generate bispecific single chain antibody molecules comprising theaforementioned CEAI specificity and the deimmunized anti-CD3 (SEQ IDNO.77) specificity firstly the variable regions of CEAI obtained by genesynthesis according to standard protocols had to be modified by PCR toobtain the corresponding single chain Fv antibody fragment. To this enda two-step fusion PCR was used to amplify the sequence coding for thevariable regions. A set of appropriate primers was designed to performthe PCR-based cloning steps, finally resulting in a single chainantibody connecting the two variable domains with a 15 amino acid linker([Gly₄Ser]₃) in the order VL-Linker-VH.

In short the following primer combinations were used:

PCR PCR Resulting step Used primers step Used primers scFv 1 5′CEAI LH +−> Fusion 5′CEAI LH + CEAI LH 3′CEAI VL Linker PCR 3′CEAI LH 2 5′CEAI VHLinker + −> 3′CEAI LH

The nucleotide sequences of the oligonucleotide primers are given below:

5′CEAI LH: (SEQ ID NO. 137) 5′ AGGTGTACACTCCGACATTGAGCTCACCCAG 3′3′CEAI VL Linker: (SEQ ID NO. 138) 5′GGAGCCGCCGCCGCCAGAACCACCACCACCTTTGATCTCGAGCTTG G 3′ 5′CEAI VH Linker:(SEQ ID NO. 139) 5′ GGCGGCGGCGGCTCCGGTGGTGGTGGTTCTCAGGTCCAACTGCAGG AG 3′3′CEAI LH: (SEQ ID NO. 140) 5′ AATCCGGAGGAGACGGTGACCG 3′

To generate the single chain antibody, two PCRs with the respectiveprimer combinations described above as PCR step 1 and 2 were performed.During this PCR overlapping complementary sequences were introduced intothe PCR-products (stemming from the respective linker primers) thatcombined to form the coding sequence of the 15 amino acid linker duringthe subsequent fusion PCR. Subsequently the amplified VH and VL domainswere joined in this fusion PCR in which only the outer primers and bothPCR-products were required. The resulting scFv antibody is flanked atthe 5′ end with the restriction enzyme recognition site for BsrGI and atthe 3′ end with the restriction enzyme recognition site for BspEI.Addition of the BsrGI site was performed as to allow for the in framefusion with the coding sequence of a murine immunoglobulin leaderpeptide as described in WO2005/040220. The BspEI site was created as toallow for the in frame fusion with the sequence coding for the CD3specific single chain antibody to generate the bispecific single chainantibody. To accomplish the fusion of the single chain Fv antibodies andto allow for eukaryotic expression the coding sequence of the CEAspecific single chain Fv antibody was cloned via BsrGI and BspEI intothe pEFDHFR expression vector (pEFDHFR was described in Mack et al.Proc. Natl. Acad. Sci. USA 92 (1995) 7021-7025) containing thedeimmunized anti-CD3 single chain Fv antibody as described inWO2005/040220; in the present invention referred to as SEQ ID NO.77.Single clones of the construct were isolated and sequenced with primerscomplementary to flanking regions in the expression vector according tostandard protocols (Sambrock, Molecular Cloning; A Laboratory Manual,2nd edition, Cold Spring Harbour laboratory Press, Cold Spring Harbour,N.Y. (1989)). For further experiments a clone of the construct with averified nucleotide sequence was selected.

2. Expression and Purification of the CEA×CD3 Bispecific Single ChainAntibodies

The bispecific single chain antibodies were expressed in chinese hamsterovary (CHO) cells. Eukaryotic protein expression in DHFR deficient CHOcells was performed as described in Kaufmann (loc. cit.). Geneamplification of the constructs were induced by increasingconcentrations of MTX to a final concentration of up to 20 nM MTX. Aftertwo passages of stationary culture the cells were grown in rollerbottles with CHO modified DMEM medium (HiQ®, HiClone) for 7 days beforeharvest. The cells were removed by centrifugation and the supernatantcontaining the expressed protein was stored at −20° C.

Äkta® FPLC System (Pharmacia) and Unicorn® Software were used forchromatography. All chemicals were of research grade and purchased fromSigma (Deisenhofen) or Merck (Darmstadt). Immobilized metal affinitychromatography (“IMAC”) was performed using a Fractogel® column (Merck)which was loaded with ZnCl₂ according to the protocol provided by themanufacturer. The column was equilibrated with buffer A2 (20 mM sodiumphosphate buffer pH 7.5, 0.4 M NaCl) and the cell culture supernatant(500 ml) was applied to the column (10 ml) at a flow rate of 3 ml/min.The column was washed with buffer A2 to remove unbound sample. Boundprotein was eluted using a 2 step gradient of buffer B2 (20 mM sodiumphosphate buffer pH 7.5, 0.4 M NaCl, 0.5 M Imidazol) according to thefollowing:

Step 1: 20% buffer B2 in 6 column volumes;

Step 2: 100% buffer B2 in 6 column volumes.

Eluted protein fractions from step 2 were pooled for furtherpurification.

Gel filtration chromatography was performed on a Sephadex S200 HiPrepcolumn (Pharmacia) equilibrated with PBS (Gibco). Eluted protein samples(flow rate 1 ml/min) were subjected to standard SDS-PAGE and WesternBlot for detection. Prior to purification, the column was calibrated formolecular weight determination (molecular weight marker kit, Sigma MWGF-200). Protein concentrations were determined using protein assay dye(MicroBCA, Pierce) and IgG (Biorad) as standard protein.

The CEA×CD3 bispecific single chain antibodies were isolated in a twostep purification process of IMAC and gel filtration. The main producthad a molecular weight of ca. 52 kDa under native conditions asdetermined by gel filtration in PBS. This molecular weight correspondsto the bispecific single chain antibody. All constructs were purifiedaccording to this method.

Purified bispecific single chain antibody protein was analyzed in SDSPAGE under reducing conditions performed with pre-cast 4-12% Bis Trisgels (Invitrogen). Sample preparation and application were performedaccording to the protocol provided by the manufacturer. The molecularweight was determined with MultiMark protein standard (Invitrogen). Thegel was stained with colloidal Coomassie (Invitrogen protocol). Thepurity of the isolated protein was >95% as determined by SDS-PAGE.

Western Blot was performed using an Optitran® BA-S83 membrane and theInvitrogen Blot Module according to the protocol provided by themanufacturer. The antibodies used were directed against the His Tag(Penta His, Qiagen) and Goat-anti-mouse Ig labeled with alkalinephosphatase (AP) (Sigma), and BCIP/NBT (Sigma) as substrate. Thebispecific single chain antibody could be specifically detected byWestern Blot. A single band was detected at 52 kD corresponding to thepurified bispecific single chain antibody molecule.

3. Flow Cytometric Binding Analysis of the CEA×CD3 Bispecific SingleChain Antibodies

In order to test the functionality of the constructs with regard tobinding capability to membrane-bound human CEA and human CD3, FACSanalysis was performed. For this purpose, human CEA-transfected CHOcells and CD3 positive human T cell leukemia cell line HPB-All (DSMZ,Braunschweig, ACC483) were used. 200,000 CEA positive CHO cells or200,000 HPB-All cells were incubated for 30 min on ice with 50 μl of thepure cell supernatant of CHO cell cultures each expressing bispecificantibodies with different arrangements of VH and VL regions of CEA andCD3 (as described above). The cells were washed twice in PBS and bindingof the construct was detected with an unlabeled murine Penta Hisantibody (diluted 1:20 in 50 μl PBS with 2% FCS; Qiagen), whichspecifically binds to cell-bound construct via the construct'sC-terminal histidine tag. A washing step followed to remove unboundmurine Penta His antibody. Bound anti-His antibodies were detected withan Fc gamma-specific antibody (Dianova) conjugated to phycoerythrin,diluted 1:100 in 50 μl PBS with 2% FCS. As a positive control forbinding to human CEA, monoclonal antibody Col-1 (see Example 3) has beenused. For control of binding to human CD3, a CD19×CD3 bispecific singlechain construct as described in WO 99/054440 has been utilized. As anegative control fresh culture medium was used in place of culturesupernatant.

Cells were analyzed by flow cytometry (FACS-Calibur; Becton Dickinson,Heidelberg). FACS staining and measuring of the fluorescence intensitywere performed as described in Current Protocols in Immunology (Coligan,Kruisbeek, Margulies, Shevach and Strober, Wiley-Interscience, 2002).

As shown in FIG. 1, several domain arrangements of the bispecific singlechain antibodies, i.e. CEAI VHVL×SEQ ID NO.77 VHVL (SEQ ID NO. 8), CEAIVLVH×SEQ ID NO.77 VHVL (SEQ ID NO. 6), CEAII VHVL×SEQ ID NO.77 VHVL (SEQID NO. 10), CEAIII VLVH×SEQ ID NO.77 VHVL (SEQ ID NO. 12) and CEAIIIVHVL×SEQ ID NO.77 VHVL (SEQ ID NO. 14), bound to human membrane-boundCEA and human CD3. As a negative control, culture medium and 1. and 2.detection antibodies have been used.

EXAMPLE 3: BINDING OF CEA×CD3 BISPECIFIC SINGLE CHAIN ANTIBODIES TOSOLUBLE HUMAN CEA

In order to determine the specificity against soluble human CEA, variousCEA×CD3 bispecific single chain antibodies were tested in ELISA.

To this end, soluble human CEA antigen was first biotinylated.Biotinylation was accomplished in PBS containing 5% DMSO (Sigma) with afifteen-fold molar excess of EZ-Link Sulfo NHS-LC-LC-Biotin (Pierce) for1 hour at room temperature in a sample mixer (Dynal). For the separationof free Biotin and biotinylated CEA antigen, the assay was excessivelydialyzed against PBS according to standard protocols. The retainedbioactivity of the biotin-labeled CEA was confirmed in ELISA bindingexperiments.

The direct ELISA to determine the specificity of CEA×CD3 bispecificsingle chain antibody against soluble human CEA was carried outaccording to standard procedures. Briefly, 50 μl/well PBS or solublebiotinylated human CEA (Abcam; 5 μg/ml in 1×PBS) were immobilized on a96-well streptavidin-coated ELISA plate (Nunc) by incubating at 4° C.for about 16 hours. After washing with 200 μl water per well 200 μl ofblocking solution (PBS/3% BSA) was added. After blocking for 1 h at roomtemperature the blocking solution was removed. All subsequent washing(200 μl/well of 1×PBS/0.05% (v/v) Tween20) and incubation steps wereperformed at room temperature. After washing once, the CEAI VHVL×SEQ IDNO.77 VHVL (SEQ ID NO. 8; anti-CEA part derived from mAb A5B7), CEAIIVHVL×SEQ ID NO.77 VHVL (SEQ ID NO. 10; anti-CEA part derived from mAbT84.66), and CEAIII VHVL×SEQ ID NO.77 VHVL (SEQ ID NO. 14; anti-CEA partderived from mAb MFE-23) bispecific single chain antibodies, and mousemonoclonal antibody CEA/CD66 Ab-3 (Col-1; Dunn) were incubated indifferent concentrations (0.5 μg/ml and 5 μg/ml in 1×PBS; 50 μl/well)for 1 hour. 1×PBS (50 μl/well) was added as a control for unspecificbinding. 3 washing steps were followed by adding 50 μl/well Penta-HisIgG (Qiagen; 2 μg/ml in 1×PBS) for detection of the His-taggedbispecific single chain antibodies. Subsequently, the wells were washed3 times and incubated with 50 μl of a horseradish peroxidase-labelledgoat anti-mouse Fc gamma-specific antibody (Jackson ImmuneResearch;1:1000 in 1×PBS) for 1 hour. After washing 3 times the ELISA wasdeveloped by adding ABTS substrate solution (Roche) and the absorbancewas measured at a wavelength of 405 nm.

FIG. 2 shows the absorbance of the different bispecific single chainantibodies detected in the ELISA. The CEAI VHVL×SEQ ID NO.77 VHVL (SEQID NO. 8; anti-CEA part derived from mAb A5B7), CEAII VHVL×SEQ ID NO.77VHVL (SEQ ID NO. 10; anti-CEA part derived from mAb T84.66), and CEAIIIVHVL×SEQ ID NO.77 VHVL (SEQ ID NO. 14; anti-CEA part derived from mAbMFE-23) bispecific single chain antibodies and the mouse monoclonalantibody Col-1 specifically bound to soluble human CEA. No bindingsignal was observed in the absence of the CEA antigen (PBS control). Insummary, the anti-CEA binding domains derived from mAbs A5B7, T84.66 andMFE-23 bind to both soluble and membrane-bound CEA.

EXAMPLE 4: BIOACTIVITY OF CEA×CD3 BISPECIFIC SINGLE CHAIN ANTIBODIES

Bioactivity of the generated CEA×CD3 bispecific single chain antibodieswas analyzed by in vitro chromium release cytotoxicity assays using thehuman gastric carcinoma cell line Kato III or the human CEA-transfectedCHO cells as target cells and stimulated human CD8 positive T cells ornative PBMC as effector cells, respectively.

The generation of the stimulated CD8+ T cells was performed as follows:

A petri dish (145 mm diameter, greiner bio-one) was precoated with ananti-CD3 antibody (OKT3 Janssen-Cilag GmbH, Orthoclone 1 mg/ml; finalconcentration 1 μg/ml) and an anti-CD28 antibody (BD, 1 mg/ml; finalconcentration 1 μg/ml) for 1 hour at 37° C. After the incubation period,the unbound protein was removed by one washing step with PBS. The freshPBMC's were isolated from peripheral blood (30-50 ml) by Ficoll gradientcentrifugation according to standard protocols. 3-5×10⁷ PBMCs were addedto the precoated petri dish in 150 ml of RPM′ 1640/10% FCS/IL-2 20 U/ml(Proleukin, Chiron) and stimulated for 2 days. At the third day thecells were collected, washed once with RPMI 1640 and transferred to alarge T-flask. IL-2 was added to a final concentration of 20 U/ml andcultivated again for one day. The CD8+ CTLs were isolated with help ofthe CD8 negative isolation kit (Dynal Biotech) by following theinstructions of the manual. The native PBMC's were used directly afterthe Ficoll gradient centrifugation without the stimulation procedure.Target cells were washed twice with PBS and labeled with 11.1 MBq ⁵¹Crin a final volume of 100 μl RPMI with 50% FCS for 45 minutes at 37° C.Subsequently, the labeled target cells were washed 3 times with 5 mlRPMI and then used in the cytotoxicity assay. The assay was performed ina 96 round bottom plate in a total volume of 250 μl supplemented RPMI(as above) with an E:T ratio of 10:1 corresponding to 5000 target cellsand 50000 effector cells per well. For the evaluation of the constructsa starting concentration of 1 μg/ml of the bispecific single chainmolecules in the assay volume and 12 threefold dilutions thereof wereapplied. The assay time was 18 hours and cytotoxicity was measured asrelative values of released chromium in the supernatant related to thedifference of maximum lysis (addition of Triton-X) and spontaneous lysis(without effector cells). All measurements were done in triplicates.Measurement of chromium activity in the supernatants was performed witha Wizard 3 gammacounter (Perkin Elmer Life Sciences GmbH, Köhn,Germany). Analysis of the experimental data was performed with Prism 4for Windows (version 4.02, GraphPad Software Inc., San Diego, Calif.,USA). Sigmoidal dose response curves typically had R² values >0.90 asdetermined by the software. EC₅₀ values calculated by the analysisprogram were used for comparison of bioactivity.

FIG. 3 shows cytotoxic activity against human CEA-transfected targetcells (CHO-CEA⁺ cells) for various domain arrangements, i.e. for SEQ IDNO.77 VHVL×CEAI VHVL (SEQ ID NO. 4) and SEQ ID NO.77 VHVL×CEAI VLVH (SEQID NO. 2) (both constructs with anti-CD3 part N-terminally), as well asfor CEAI VLVH×SEQ ID NO.77 VHVL (SEQ ID NO. 6) and CEAI VHVL×SEQ IDNO.77 VHVL (SEQ ID NO. 8) (anti-CD3 C-terminally). Non-transfected CHOcells (lacking human CEA) were used as a negative control.

In FIG. 4, CEAI VHVL×SEQ ID NO.77 VHVL (SEQ ID NO. 8), CEAIII VLVH×SEQID NO.77 VHVL (SEQ ID NO. 12), CEAIII VHVL×SEQ ID NO.77 VHVL (SEQ ID NO.14), and CEAII VHVL×SEQ ID NO.77 VHVL (SEQ ID NO. 10) exhibitedcytotoxic activity against human CEA-transfected CHO cells.Non-transfected CHO cells (lacking human CEA) were used as a negativecontrol. As set forth above, CEAI denotes a variable region derived frommurine mAb A5B7, CEAII is a variable region derived from murine mAbT84.66 and CEAIII refers to a variable region from murine mAb MFE-23.

In summary, all tested constructs showed cytotoxic activity against(human) CEA expressing Kato III and human CEA-transfected CHO cells, inthe absence of soluble human CEA.

EXAMPLE 5: BIOACTIVITY OF CEA×CD3 BISPECIFIC SINGLE CHAIN ANTIBODIESSPECIFIC IN THE PRESENCE OF SOLUBLE CEA ANTIGEN

The competition assays are performed as described in Example 4, with thefollowing exception: The bioactivity of the CEA×CD3 bispecific singlechain antibodies is tested in the presence of various concentrations ofsoluble human CEA antigen. The soluble human CEA antigen (AbCAM Ltd.Cambridge UK) used was isolated from a metastatic colonic carcinoma fromthe liver of a single patient. Experimentally, the competition assay wascarried out by pre-incubation of a given amount of the bispecific singlechain antibody with increasing amounts of soluble human CEA antigen(either 0 μg/ml; 0.1 μg/ml; 1 μg/ml, or 0 μg/ml; 0.004 μg/ml; 0.02 μg/m;0.1 μg/ml; 0.5 μg/ml; 1 μg/ml) for 30 minutes at 37° C. prior to theaddition of the cells. The remainder of the assay was carried out asdescribed in Example 4. The results of these competition experiments areshown in FIGS. 5 to 11.

Cytotoxic activity of bispecific single chain antibodies CEAII VHVL×SEQID NO.77 VHVL (SEQ ID NO. 10; see FIGS. 7 and 9 to 11) with the anti-CEApart derived from mAb T84.66, and CEAIII VLVH×SEQ ID NO.77 VHVL (SEQ IDNO. 12; see FIG. 8) with the anti-CEA region derived from mAb MFE-23,against human CEA-positive target cells was drastically inhibited byincreasing amounts of soluble human CEA antigen. As set forth above,said constructs bind to both membrane-bound and soluble human CEAantigen; see e.g. Examples 2 and 3 and FIGS. 1 and 2. Thus, mostprobably, soluble human CEA prevents the anti-CEA part of saidbispecific single chain antibodies from binding to membrane-bound humanCEA on the target cells, for instance CHO-CEA+ or Kato III tumor cells,thereby inhibiting the cytotoxic activity mediated by said antibodyconstructs.

In contrast, it has been surprisingly found that bispecific single chainantibodies with an anti-CEA part derived from mAb A5B7 are resistant tosoluble human CEA: For instance, the cytotoxic activity mediated by CEAIVLVH×SEQ ID NO.77 VHVL (SEQ ID NO. 6; see FIG. 5), CEAI VHVL×SEQ IDNO.77 VHVL (SEQ ID NO. 8; see FIGS. 5, 8 and 10), SEQ ID NO.77 VHVL×CEAIVHVL (SEQ ID NO. 4; see FIG. 6) and SEQ ID NO.77 VHVL×CEAI VLVH (SEQ IDNO. 2; see FIG. 6) is not inhibited by increasing amounts of solublehuman CEA, not even by high concentrations (1 μg/ml). This could not beexpected in view of the fact that the anti-CEA part of said bispecificsingle chain antibodies binds to soluble human CEA (FIG. 2). Rather,inhibition of cytotoxic activity against CEA-positive target cells byincreasing amounts of soluble human CEA could have been expected, as itwas the case for T84.66- and MFE-23-derived constructs; see above.

Thus, the present invention provides for pharmaceutical compositionswith cytotoxic anti-tumor activity in the presence of even high levelsof soluble CEA antigen. Therefore, these pharmaceutical compositions areparticularly suitable for the treatment of tumor patients with highsoluble CEA antigen concentrations in their plasma, such as patientswith progressive epithelial tumors, metastatic epithelial tumors, hightumor load/burden, late stage epithelial tumors or tumor patients with aCEA serum concentration higher than 100 ng/ml, as determined by ELISA.

EXAMPLE 6: SELECTION OF HUMAN VL REGIONS

In order to provide for pharmaceutical compositions with reducedimmunogenicity when being administered to cancer patients, humanbispecific single chain antibodies with resistance to soluble CEAantigen have been generated. In a first step, human VL regions withresistance to soluble CEA have been isolated. Thus, the aim of thisexperiment is the selection of human VL regions which can pair with thematernal, murine VH of monoclonal antibody (mAb) A5B7.

1. Biotinylation of Soluble Human CEA Antigen

For phage library selection, soluble CEA antigen was biotinylated.Biotinylation was accomplished in PBS containing 5% DMSO (Sigma) with afifteenfold molar excess of EZ-Link Sulfo NHS-LC-LC-Biotin (Pierce) for1 hour at room temperature in a sample mixer (Dynal). For the separationof free Biotin and biotinylated CEA antigen, the assay was excessivelydialized against PBS according to standard protocols.

The retained bioactivity of the biotin-labeled CEA was confirmed inELISA binding experiments.

2. Isolation of RNA from Human B-Cells

100 mL blood were taken from five healthy human donors. Peripheral bloodmononuclear cells (PBMCs) were isolated by a ficoll-gradient accordingto standard methods. Total RNA was isolated from the isolated cellsusing the RNeasy® Midi Kit (QIAGEN) following the manufacturer'sinstructions. cDNA was synthesized according to standard methods(Sambrook, Cold Spring Harbor Laboratory Press 1989, 2001).

3. PCR-Amplification of Variable Light Chain Regions (VL-Regions)

For the isolation of light chain V-region DNA, RT-PCR was carried outusing Vkappa-(5′-huVK1-SacI-2001 (5′-GAGCCGCACG AGCCCGAGCT CCAGATGACCCAGTCTCC-3′) (SEQ ID NO. 78), 5′-huVK2/4-SacI-2001 (5′-GAGCCGCACGAGCCCGAGCT CGTGATGACY CAGTCTCC-3′) (SEQ ID NO. 79), 5′-huVK3-SacI-2001(5′-GAGCCGCACG AGCCCGAGCT CGTGWTGACR CAGTCTCC-3′) (SEQ ID NO. 80),5′-huVK5-SacI-2001 (5′-GAGCCGCACG AGCCCGAGCT CACACTCACG CAGTCTCC-3′)(SEQ ID NO. 81), 5′-huVK6-SacI-2001 (5′-GAGCCGCACG AGCCCGAGCT CGTGCTGACTCAGTCTCC-3′) (SEQ ID NO. 82), 3′-hu-Vk-J1-SpeI-BsiWI (5′-GACGACACTAGTTGCAGCCA CCGTACGTTT GATTTCCACC TTGGTCC-3′) (SEQ ID NO. 83),3′-hu-Vk-J2/4-SpeI-BsiWI (5′-GACGACACTA GTTGCAGCCA CCGTACGTTT GATCTCCASCTTGGTCC-3′) (SEQ ID NO. 84), 3′-hu-Vk-J3-SpeI-BsiWI (5′-GACGACACTAGTTGCAGCCA CCGTACGTTT GATATCCACT TTGGTCC-3′) (SEQ ID NO. 85),3′-hu-Vk-J5-SpeI-BsiWI (5′-GACGACACTA GTTGCAGCCA CCGTACGTTT AATCTCCAGTCGTGTCC-3′) (SEQ ID NO. 86)) and V lambda (5′-huVL1a-SacI-2001 (GAG CCGCAC GAG CCC GAG CTC GTG TTG ACG CAG CCG CCC TC) (SEQ ID NO. 87),5′-huVL1b-SacI-2001 (GAG CCG CAC GAG CCC GAG CTC GTG CTG ACT CAG CCA CCCTC) (SEQ ID NO. 88), 5′-huVL2-SacI-2001 (GAG CCG CAG GAG CCC GAG CTC GCCCTG ACT CAG CCT SCC TCC GT) (SEQ ID NO. 89), 5′-huVL4-SacI-2001 (ACC TGCGAG CTC GTG CTG ACT CAR YCM YCC TCT GC) (SEQ ID NO. 90),5′-huVL5-SacI-2001 (ACC TGC GAG CTC GTG CTG ACT CAG CCR SCT TCC) (SEQ IDNO. 91), 5′-huVL6-SacI-2001 (ACC TGC GAG CTC ATG CTG ACT CAG CCC CAC TC)(SEQ ID NO. 92), 5′-huVL3/9-SacI-2001 (GAG CCG CAC GAG CCC GAG CTC GWGCTG ACT CAG CCA CCY TC) (SEQ ID NO. 93), 5′-huVL7/8-SacI-2001 (GAG CCGCAC GAG CCC GAG CTC GTG GTG ACY CAG GAG CCM TC) (SEQ ID NO. 94),3′-hu-Vlam-BlnI-SpeI-2001 (CGT GGG ACT AGT CTT GGG CTG ACC TAG GAC GGT)(SEQ ID NO. 95), 3′-hu-Vlam2-BlnI-SpeI-2002: CGT GGG ACT AGT CTT GGG CTGACC GAG GAC GGT) (SEQ ID NO. 96) primer sets.

RNA from human B-cells was transcribed into cDNA (as described above)and used as template DNA in PCR reactions. Per PCR reaction, one5′-primer was combined with one 3′-primer. The number of different PCRreactions was determined by the number of possible combinations of 5′-and 3′-primers. The following PCR-program was used for amplification:Denaturation at 94° C. for 15 seconds, primer annealing at 52° C. for 50seconds and primer extension at 72° C. for 90 seconds were performedover 40 cycles, followed by final extension at 72° C. for 10 minutes.Light chain DNA V-fragments were then isolated according to standardprotocols.

4. Library Construction—Cloning of the Human VL Pool

A phage display library was generally constructed based on standardprocedures, as for example disclosed in “Phage Display: A LaboratoryManual”; Ed. Barbas, Burton, Scott & Silverman; Cold Spring HarborLaboratory Press, 2001.

The primers chosen for PCR amplification gave rise to 5′-SacI and3′-SpeI recognition sites for the light chain V-fragments. Four ligationreactions were set up, each consisting of 400 ng of light chainfragments (SacI-SpeI digested, 2×kappa and 2×lambda) and 1400 ng of thephagemid pComb3H5BHis (SacI-SpeI digested; large fragment; this vectoris described in the thesis dissertation of Dr. Ralf Lutterbüse. The fourresulting antibody V-light chain pools were then each transformed into300 μL of electrocompetent Escherichia coli XL1 Blue by electroporation(2.5 kV, 0.2 cm gap cuvette, 25 mF, 200 Ohm, Biorad gene-pulser)resulting in library sizes of

kappa1: 2 × 10⁸ kappa2: 6 × 10⁷ lambda1: 9 × 10⁷ lambda2: 6 × 10⁷independent clones.

Kappa (light chain) DNA-fragments from the different PCR amplificationswere weighted for each ligation as follows: Each 5′-primer defines aspecific group. Within these groups the 3′-primers define the subgroups.The kappa subgroups were weighted 1:2:1:1 corresponding to the primers3′-hu-Vk-J1-SpeI-BsiWI: 3′-hu-Vk-J2/4-SpeI-BsiWI:3′-hu-Vk-J3-SpeI-BsiWI: 3′-hu-Vk-J5-SpeI-BsiWI. The groups were weightedaccording to their germline distribution 1:1:1:0.2:0.2 corresponding tothe primers5′-huVK1-Sac-2001:5′-huVK3-Sac-2001:5′-huVK2/4-Sac-2001:5′-huVK5-Sac-2001:5′-huVK6-Sac-2001.

Lambda (light chain) DNA-fragments from the different PCR amplificationswere weighted for each ligation as follows: Each 5′-primer defines aspecific group. Within these groups the 3′-primers define the subgroups.The lambda subgroups were weighted 3:1 corresponding to the primers3′-hu-Vlam-BlnI-SpeI-2001: 3′-hu-Vlam2-BlnI-SpeI-2002.

The groups were weighted according to their germline distribution1:1:2:2:2:3 corresponding to the primers5′-huVL1a-SacI-2001:5′-huVL1b-SacI-2001:5′-huVL2-SacI-2001:5′-huVL4-SacI-2001+5′-huVL5-SacI-2001:5′-huVL6-SacI-2001+5′-huVL7/8-SacI-2001:5′-huVL3/9-SacI-2001.

After electroporation each reaction was incubated in SOC broth (Fluke)for phenotype expression. The two kappa cultures were combined as wellas the two lambda cultures. The resulting kappa culture and theresulting lambda culture were then each incubated in 500 mL of SBselection medium containing 50 μg/mL carbenicillin and 2% w/v glucoseovernight. The next day, cells were harvested by centrifugation andplasmid preparation was carried out using a commercially availableplasmid preparation kit (Qiagen).

5. Construction of the Antibody Library—Human VL—Maternal VH

PCR was performed to amplify the maternal VH of mAb A5B7 from a vectorcontaining said maternal VH. For amplification a PCR protocol accordingto standard procedures was followed using the 5′-primer 5′-AVH-XhoI(5′-GTC ACA CTC GAG TCA GGA GGA GGC TTG GTA C-3′) (SEQ ID NO. 97) andthe 3′-primer 3′-AVH-BstEII (5′-GTC ACA GGT GAC CGT GGT CCC TTG GCC CCAG-3′ (SEQ ID NO. 98). After purification of the approximately 350 bpamplification product from an analytical agarose gel, the DNA fragmentwas cut with the restriction enzymes BstEII and XhoI. The phagemidpComb3H5BHis (this vector is described in the thesis dissertation of Dr.Ralf Lutterbüse) was digested accordingly and the large fragment wasligated with the above mentioned fragment. After transformation into E.coli XL1 blue, a single clone was cultivated in 100 mL SB medium(containing 50 μg/mL carbenicilline) and the plasmid was preparedaccording to standard protocols. The successful cloning was confirmed bysequencing the insert (Sequiserve, Munich).

This vector pComb3H5BHis/maternalVH of mAb A5B7 was restricted with therestriction enzymes SacI and SpeI. The large vector fragment wasisolated. Plasmid-DNA containing the Vkappa- and the Vlambda library wasrestricted with the restriction enzymes SacI and SpeI. The smallVkappa—and the respective Vlambda fragment (each approximately 350 bp)were isolated according to standard protocols. 1200 ng of the vectorfragment were ligated with a mix of each 200 ng of both the Vkappa andthe Vlambda fragments. The ligation reaction was transformed into 300 μLof electrocompetent E. coli XL1 Blue by electroporation (2.5 kV, 0.2 cmgap cuvette, 25 mF, 200 Ohm) resulting in a total scFv library size of1.2×10⁸ independent clones.

After phenotype expression and slow adaptation to carbenicillin, theantibody library was transferred into SB-Carbenicillin (50 μg/mL)selection medium. The antibody library was then infected with aninfectious dose of 1×10¹² particles of helper phage VCSM13 resulting inthe production and secretion of filamentous M13 phage, wherein eachphage particle contained single stranded pComb3H5BHis-DNA encoding ahalf-human scFv-fragment and displayed the corresponding scFv-protein asa translational fusion to phage coat protein III.

6. Phage Display Selection of a Human VL

The phage particles carrying the scFv-repertoire were harvested from theculture supernatant by PEG8000/NaCl precipitation and centrifugation.Then approximately 1×10¹¹ to 1×10¹² scFv phage particles wereresuspended in 0.5 mL of TBS/1% BSA and incubated with biotinylatedsoluble CEA, that was immobilized in Streptavidine coated wells of anELISA plate (Nunc) for 1 h. A 10 μg antigen/ml PBS solution (50 μl) wasincubated for over night at 4° C. in the streptavidine coated wells,washed once with water, followed by blocking for 1 hour at 37° C. with200 μl of 3% BSA in TBS, that was removed after incubation.

scFv phage that did not specifically bind to the target antigen wereeliminated by washing steps with TBS/0.05% Tween. This washing procedurewas repeated up to 10 times in further rounds.

After washing, binding entities were eluted by using HCl-glycine, pH2.2. Following neutralization with 2 M Tris, pH 12, the eluate was usedfor infection of a fresh uninfected E. coli XL1 Blue culture.

To elute remaining high binding entities 50 μL of a fresh E. coli XL1blue culture (OD600≥0.5) were added to the wells and incubated for 15minutes. Both cultures were then mixed and cells successfully transducedwith a phagemid copy, encoding a human scFv-fragment, were againselected for carbenicillin resistance and subsequently infected withVCMS13 helper phage to start the second round of antibody display and invitro selection.

Plasmid DNA corresponding to 4 rounds of panning was isolated from E.coli cultures. For the production of soluble scFv-protein, VH-VL-DNAfragments were excised from the plasmids (XhoI-SpeI), and cloned via thesame restriction sites in the plasmid pComb3H5BFlag/His, in which theexpression construct (e.g. scFv) includes a Flag-tag (TGDYKDDDDK) (SEQID NO. 99) between the scFv and the His6-tag and the additional phageproteins are deleted.

After ligation each pool (different rounds of panning) of plasmid DNAwas transformed into 100 μL heat shock competent E. coli TG1 and platedonto carbenicillin LB-agar. Single colonies were picked and inoculatedinto 120 μL of LB carb (50 μg/mL) 1% glucose in 96-well plates(Greiner). The wells were sealed with a semipermeable membrane (Greiner)and the plates were incubated overnight at 37° C. in a shaking incubator(master plate). Then 10 μL of the master plate cultures were transferredinto a second 96 well plate (working plate) containing 90 μL LB Garb (50μg/mL) 0.1% glucose per well. After incubation for 4 h in a 37° C.shaking incubator, scFv production was induced by adding 20 μL LB carb 6mM IPTG to each well. After another incubation step overnight at 30° C.with shaking, cells were lysed in a 1 h incubation at room temperaturewith 40 μL lysis buffer (400 mM boric acid, 320 mM NaCl, 4 mM EDTA pH 8,2.5 mg/mL lysozyme). Residual cells and cell debris were separated bycentrifugation for 12 minutes at 1,900×g (Hettich).

The supernatants containing scFv molecules were then tested for bindingin flow cytometric binding assays. CHO cells transfected with human CEAwere used as CEA-positive cell line. Cell binding assays were carriedout by initially incubating between 100,000 and 200,000 cells withperiplasmic preparation containing human scFv or relevant controls.After incubation the cells were washed in PBS/1% FCS (fetal calf serum)and further incubated with 5-10 μg/ml of anti-FLAG M2 antibody (Sigma).After the cells had again been washed, they were incubated withpolyclonal, PE-labeled anti-mouse antibodies (Dianova) and subsequentlyanalyzed by flow cytometry. Approximately 600 clones were tested forbinding signals on CEA-positive CHO cells. 27 positive clones wereobtained. After sequencing of the respective scFv DNA, a total of 9different sequences were obtained.

FIG. 12 depicts binding of the nine different half-human scFv (i.e.murine A5B7 VH-human VL) constructs to various cell lines as measured byflow cytometric analysis. The Figure contains multiple diagrams, one foreach construct tested. In any given diagram, the black distributionshows fluorescence intensity for cells incubated only with PBS alone inthe absence of any construct but with all appropriate detection agentsas used for detection of scFvs. In this way, any fluorescence shiftobserved can be definitely attributed to scFv construct rather thandetection agents or buffer. Shifts in fluorescence which are indicativeof construct binding to the respective cell line are depicted by a grayline in each diagram. Generally, a shift of higher magnitude away from,i.e. further to the (black) control indicates stronger binding, whereasa shift of lower magnitude away from, i.e. closer to the (black) controlindicates weaker binding.

It can be seen from FIG. 12 that the constructs A-121, A-183, A-240,A-313, A-290, A-315, A4-35, A4-52, MP2-A5 show clearly discernableshifts in fluorescence intensity as compared to the respective control,indicative of binding of the scFvs to membrane-bound CEA on the CHOtarget cells. In the following, the human VL region of scFv A-240 (SEQID NO. 48) has been selected and used for the isolation of a human VHregion. Said human VL region is depicted in SEQ ID NOs. 63 (nucleotidesequence) and 64 (amino acid sequence).

EXAMPLE 7: CONSTRUCTION OF THE ANTIBODY LIBRARIES AND PHAGE DISPLAYSELECTION OF HUMAN VH REGIONS RESISTANT TO SOLUBLE CEA ANTIGEN

The aim of the following experiments is the selection of a set of humanVH regions resistant to soluble CEA antigen that pair with the human VLregion of scFv A-240, selected as described in Example 6. Said human VLregion is depicted in SEQ ID NOs. 63 (nucleotide sequence) and 64 (aminoacid sequence).

1. Isolation of RNA from Peripheric Blood Mononuclear Cells (PBMCs)

100 mL blood were taken from five healthy human donors. Peripheral bloodmononuclear cells (PBMCs) were isolated by a ficoll-gradient accordingto standard methods. Total RNA was isolated from PBMCs using the RNeasy®Midi Kit (QIAGEN) following the manufacturer's instructions. cDNA wassynthesized according to standard methods (Sambrook, Cold Spring HarborLaboratory Press 1989, 2001).

2. PCR-Amplification of Variable Heavy Chain Regions (VH-Regions)

The VH library was constructed and named Lib 134-VH. This VH-libraryconsists of the human repertoire of FR1-CDR1-FR2-CDR2-FR3 from the PCRamplified VH-regions of the above described PBMC pool, linkedoperatively to the VH CDR3 of the maternal antibody followed by a humanFR4 germline sequence.

For the isolation of human template VH-regions, RT-PCR was carried outusing a 5′-VH-specific primer set (5′-huVH1,3,5-XhoI-2001 (5′-AGG TGCAGC TGC TCG AGT CTG G-3′) (SEQ ID NO. 100), 5′-huVH4-XhoI-2001 (5′-CAGGTG CAG CTG CTC GAG TCG GG-3′) (SEQ ID NO. 101), 5′-huVH4B-XhoI-2001(5′-CAG GTG CAG CTA CTC GAG TGG GG-3′) (SEQ ID NO. 102)) and a set oftwo 3′-VH-specific primers (3′-hu-VH-BstEII-2001 (5′-CTG AGG AGA CGG TGACC-3′) (SEQ ID NO. 103), 3′-hu-VH-J3-BstEII-2001 (5′-CTG AAG AGA CGG TGACC-3′) (SEQ ID NO. 104)). Per PCR reaction, one 5′-primer was combinedwith one 3′-primer; the number of different PCR reactions was determinedby the number of possible combinations of 5′- and 3′-primers. The PBMCcDNA of five donors was used as a source of VH-genes. The followingPCR-program was used for amplification: Denaturation at 94° C. for 15seconds, primer annealing at 52° C. for 50 seconds and primer extensionat 72° C. for 60 seconds was performed over 40 cycles, followed by finalextension at 72° C. for 10 minutes. The amplification products with asize of approximately 350 bp were isolated according to standardmethods.

For the isolation of Lib 134-VH-regions, RT-PCR was carried out in twosteps. First, the human heavy chain VH-segments (FR1-CDR1-FR2-CDR2-FR3)were PCR-amplified from the isolated template VH fragments using thesame 5′-VH-specific primer set as described above(5′-huVH1,3,5-XhoI-2001, 5′-huVH4-XhoI-2001, 5′-huVH4B-XhoI-2001; SEQ IDNOs. 100-102) and a 3′-specific primer set (3′-A134-VH1A (5′-GTA GTC AAAGTA GAA CCG TAG CCC CCT ATC TCT YGC ACA GTA ATA CAC GGC—3′) (SEQ ID NO.105), 3′-A134-VH1B (5′-GTA GTC AAA GTA GAA CCG TAG CCC CCT ATC TCT YGCACA GTA ATA CAY RGC—3′) (SEQ ID NO. 106), 3′-A134-VH3A (5′-GTA GTC AAAGTA GAA CCG TAG CCC CCT ATC TCT TGY ACA GTA ATA CAC RGC—3′) (SEQ ID NO.107), wherein the indicated “T” may also be replaced by “A”, “C” or “G”,3′-A134-VH3B (5′-GTA GTC AAA GTA GAA CCG TAG CCC CCT ATC TCT TGC ACA GTAATA CAA RGC—3′) (SEQ ID NO. 108), wherein the indicated “T” may also bereplaced by “A”, “C” or “G”, 3′-A134-VH4 (5′-GTA GTC AAA GTA GAA CCG TAGCCC CCT ATC TCT SGC ACA GTA ATA CAC RGC—3′) (SEQ ID NO. 109)) for thehuman VH subfamilies 1, 3 and 4 matching in the very terminal region ofFR3.

The following primer combinations were used:

a) 5′-huVH1,3,5-XhoI-2001×3′-A134-VH1A

b) 5′-huVH1,3,5-XhoI-2001×3′-A134-VH1B

c) 5′-huVH1,3,5-XhoI-2001×3′-A134-VH3A

d) 5′-huVH1,3,5-XhoI-2001×3′-A134-VH3B

e) 5′-huVH4-XhoI-2001×3′-A134-VH4

f) 5′-huVH4B-XhoI-2001×3′-A134-VH4

Per PCR reaction, one 5′-primer was combined with the 3′-primer; thenumber of different PCR reactions was determined by the number ofpossible combinations of 5′- and the 3′-primer. The followingPCR-program was used for amplification: Denaturation at 94° C. for 15seconds, primer annealing at 52° C. for 50 seconds and primer extensionat 72° C. for 90 seconds was performed over 40 cycles, followed by finalextension at 72° C. for 10 minutes. Through this PCR step and therespective 3′-primer sequence, the human VH segments are prolonged for apart of the maternal VH CDR3, which then in turn is the priming site forthe second step PCR 3′-primer.

These VH-(FR1-CDR1-FR2-CDR2-FR3) DNA-fragments were then used astemplates in a second PCR reaction using again the respective5′VH-specific primer and a universal 3′ primer matching to the universal3′-terminus of the amplified DNA-fragments (3′ A134-JH6-BstEII, 5′-CGAGAC GGT GAC CGT GGT CCC TTG GCC CCA GTA GTC AAA GTA GAA CCG TAG CC—3′)(SEQ ID NO. 110).

The following PCR-program was used for amplification:

Denaturation at 94° C. for 15 seconds, primer annealing at 52° C. for 50seconds and primer extension at 72° C. for 60 seconds were performedover 40 cycles, followed by final extension at 72° C. for 10 minutes.The DNA V-fragments were isolated according to standard protocols.

3. Library Construction—Cloning of the Human VH Pool

In a second round of the foregoing method, the human VL of scFv A-240identified in the first, previous selection (see Example 6) was chosen,and subsequently combined with the library of human VH fragments withthe aim of generating a human scFv. A phage display library wasgenerally constructed based on standard procedures, as for exampledisclosed in “Phage Display: A Laboratory Manual”; Ed. Barbas, Burton,Scott & Silverman; Cold Spring Harbor Laboratory Press, 2001.

Heavy chain DNA-fragments from the different PCR amplifications wereweighted for each ligation as follows:

a:b:c:d:e:f=3:1:3:1:1:1, wherein a-f have the following meanings:

a) 5′-huVH1,3,5-XhoI-2001×3′-A134-VH1A

b) 5′-huVH1,3,5-XhoI-2001×3′-A134-VH1B

c) 5′-huVH1,3,5-XhoI-2001×3′-A134-VH3A

d) 5′-huVH1,3,5-XhoI-2001×3′-A134-VH3B

e) 5′-huVH4-XhoI-2001×3′-A134-VH4

f) 5′-huVH4B-XhoI-2001×3′-A134-VH4

One ligation reaction was set up consisting of 400 ng of human Lib134-VH fragment pool (XhoI-BstEII digested) and 1200 ng of the plasmidpComb3H5BHis/A-240 VL (the DNA encoding the VL region of scFv A-240 wascloned via the restriction sites SacI and SpeI into pComb3H5BHisaccording to standard procedures). The resulting antibody human VH poolwas then transformed into 300 μL of electrocompetent Escherichia coliXL1 Blue by electroporation (2.5 kV, 0.2 cm gap cuvette, 25 mF, 200 Ohm,Biorad gene-pulser) resulting in a library size of 0.8×10⁸ independentclones in total.

After electroporation the assay was incubated in SOC broth (Fluka) forphenotype expression. The cultures were then each incubated in 500 mL ofSB selection medium containing 50 μg/mL carbenicillin and 2% v/v glucoseovernight. The next day, cells of the cultures were harvested bycentrifugation and plasmid preparation was carried out using acommercially available plasmid preparation kit (Qiagen) to preserve theDNA library.

1.5 μg of this plasmid pool encoding the respective scFv pool were thenelectroporated into E. coli XL1blue (2.5 kV, 0.2 cm gap cuvette, 25 mF,200 Ohm, Biorad gene-pulser) resulting in a library size of 2.4×10⁹independent clones in total. After phenotype expression and slowadaption to carbenicillin the antibody library was transferred intoSB-Carbenicillin (50 μg/mL) selection medium. The antibody library wasthen infected with an infectious dose of 1×10¹² particles of helperphage VCSM13 resulting in the production and secretion of filamentousM13 phage, wherein each phage particle contained single strandedpComb3H5BHis-DNA encoding a human scFv-fragment and displayed thecorresponding scFv-protein as a translational fusion to phage coatprotein III.

4. Phage Display Selection of a Human VH

The phage particles carrying the human scFv-repertoire were harvestedfrom the culture supernatant by PEG8000/NaCl precipitation andcentrifugation. Then approximately 1×10¹¹ to 1×10¹² scFv phage particleswere resuspended in 0.5 mL of TBS/1% BSA and incubated with biotinylatedsoluble CEA, that was immobilized in Streptavidine coated wells of anELISA plate (Nunc) for 1 h. A 10 μg antigen/ml PBS solution (50 μl) wasincubated for over night at 4° C. in the streptavidine coated wells,washed once with water, followed by blocking for 1 hour at 37° C. with200 μl of 3% BSA in TBS, that was removed after incubation.

scFv phage that did not specifically bind to the target antigen wereeliminated by washing steps with TBS/0.05% Tween. This washing procedurewas repeated up to 10 times in further rounds.

After washing, binding entities were eluted by using HCl-glycine, pH2.2. Following neutralization with 2 M Tris, pH 12, the eluate was usedfor infection of a fresh uninfected E. coli XL1 Blue culture.

To elute remaining high binding entities 50 μL of a fresh E. coli XL1blue culture (OD600≥0.5) were added to the wells and incubated for 15minutes. Both cultures were then mixed and cells successfully transducedwith a phagemid copy, encoding a human scFv-fragment, were againselected for carbenicillin resistance and subsequently infected withVCMS13 helper phage to start the second round of antibody display and invitro selection.

Plasmid DNA corresponding to 4 rounds of panning was isolated from E.coli cultures. For the production of soluble scFv-protein, VH-VL-DNAfragments were excised from the plasmids (XhoI-SpeI), and cloned via thesame restriction sites in the plasmid pComb3H5BFlag/His, in which theexpression construct (e.g. scFv) includes a Flag-tag (TGDYKDDDDK; SEQ IDNO. 99) between the scFv and the His6-tag and the additional phageproteins are deleted.

After ligation each pool (different rounds of panning) of plasmid DNAwas transformed into 100 μL heat shock competent E. coli TG1 and platedonto carbenicillin LB-agar. Single colonies were picked and inoculatedinto 120 μL of LB carb (50 μg/mL) 1% glucose in 96-well plates(Greiner). The wells were sealed with a semipermeable membrane (Greiner)and the plates were incubated overnight at 37° C. in a shaking incubator(master plate). Then 10 μL of the master plate cultures were transferredinto a second 96 well plate (working plate) containing 90 μL LB carb (50μg/mL) 0.1% glucose per well. After incubation for 4 h in a 37° C.shaking incubator, scFv production was induced by adding 20 μL LB Garb 6mM IPTG to each well. After another incubation step overnight at 30° C.with shaking, cells were lysed in a 1 h incubation at room temperaturewith 40 μL lysis buffer (400 mM boric acid, 320 mM NaCl, 4 mM EDTA pH 8,2.5 mg/mL lysozyme). Residual cells and cell debris were separated bycentrifugation for 12 minutes at 1,900×g (Hettich).

The supernatants containing scFv molecules were then tested for bindingin flow cytometric binding assays.

CHO cells transfected with human CEA were used as CEA-positive cellline. Cell binding assays were carried out by initially incubatingbetween 100,000 and 200,000 cells with periplasmic preparationcontaining human scFv or relevant controls. After incubation the cellswere washed in PBS/1% FCS (fetal calf serum) and further incubated with5-10 μg/ml of anti-FLAG M2 antibody. After the cells had again beenwashed, they were incubated with polyclonal, PE-labeled anti-mouseantibodies (Dianova) and subsequently analyzed by flow cytometry. 46clones were tested for binding signals on CEA-positive CHO cells. All ofthem showed positive signals. After sequencing of the respective scFvDNA a total of 9 different sequences were obtained, eight of whichdisplayed a high degree of homology. The human constructs MP510_3-A5.3(MP510-A5; SEQ ID NO. 50), MP510_3-B9.1 (MP511-B9; SEQ ID NO. 52),MP510_3-D8.1 (MP511-D8; SEQ ID NO. 54) have been selected for furthercharacterization. The corresponding amino acid sequences are shown inthe sequence listing.

Periplasmic extracts of said human constructs MP510-A5, MP511-B9,MP511-D8 as well as the half human construct A-240 Vlambda.3 (murine VHA5B7/human VL A240; SEQ ID NO. 48) were further analyzed in flowcytometric experiments with CEA-positive and -negative cell lines. Itcan be seen from FIG. 13, that the human constructs MP510-A5 (SEQ ID NO.50), MP511-B9 (SEQ ID NO. 52), MP511-D8 (SEQ ID NO. 54) show clearlydiscernable shifts in fluorescence intensity as compared to therespective half-human control A-240 Vlambda.3 (murine VH A5B7/human VLA240; SEQ ID NO. 48). Thus, the human scFv constructs show strongerbinding activity to membrane-bound human CEA than the half humanconstruct A-240 Vlambda.3. In addition, all of the human constructsshowed distinct binding to CEA-positive human KATO III cells (humangastric cancer cell line), whereas none of them showed binding toCEA-negative, untransfected CHO cells as well as to CEA-negative humanNALM 6 cells (human B cell line) (data not shown).

EXAMPLE 8: GENERATION AND CYTOTOXIC ACTIVITY OF HUMAN CEA×CD3 BISPECIFICSINGLE CHAIN ANTIBODIES

1. Arrangements

In the next step, various domain arrangements of human bispecific singlechain antibody molecules have been generated. These molecules comprisethe human anti-CEA binding domain (the generation of which has beendescribed in Example 7, supra) and the de-immunized binding domain withspecificity for the human CD3 antigen shown in SEQ ID NO.77 VHVL weredesigned as set out in Table 1; see also Example 2, supra. Allbispecific single chain antibody constructs with the human anti-CEAbinding domains described herein (apparently) bind to soluble human CEAantigen since they have been isolated after four rounds of phage displayselection on soluble CEA antigen immobilized on ELISA plates.

In particular, the following arrangements have been generated:

(a) human anti-CEA part located N-terminally:

(i) VH-VL orientation of anti-CEA:

A5 VH-A240 VL×SEQ ID NO.77 VHVL (SEQ ID NO. 24),

A5 VH-A240 VL#×SEQ ID NO.77 VHVL (SEQ ID NO. 126),

B9 VH-A240 VL×SEQ ID NO.77 VHVL (SEQ ID NO. 32),

B9 VH-A240 VL#×SEQ ID NO.77 VHVL (SEQ ID NO. 130),

D8 VH-A240 VL×SEQ ID NO.77 VHVL (SEQ ID NO. 40)

D8 VH-A240 VL#×SEQ ID NO.77 VHVL (SEQ ID NO. 134), and

CEAI VH-A240 VL×SEQ ID NO.77 VHVL (SEQ ID NO. 16).

(ii) VL-VH orientation of anti-CEA:

A240 VL-A5 VH×SEQ ID NO.77 VHVL (SEQ ID NO. 26),

A240 VL-B9 VH×SEQ ID NO.77 VHVL (SEQ ID NO. 34),

A240 VL-D8 VH×SEQ ID NO.77 VHVL (SEQ ID NO. 42),

and A240 VL-CEAI VH×SEQ ID NO.77 VHVL (SEQ ID NO. 18).

(b) human anti-CEA part located C-terminally:

(i) VH-VL orientation of anti-CEA:

SEQ ID NO.77 VHVL×A5 VH-A240VL (SEQ ID NO. 30),

SEQ ID NO.77 VHVL×A5 VH-A240VL# (SEQ ID NO. 128),

SEQ ID NO.77 VHVL×B9 VH-A240VL (SEQ ID NO. 36),

SEQ ID NO.77 VHVL×E12 VH-A240VL (SEQ ID NO. 143),

SEQ ID NO.77 VHVL×B9 VH-A240VL# (SEQ ID NO. 132), SEQ ID NO.77 VHVL×D8VH-A240VL (SEQ ID NO. 44),

SEQ ID NO.77 VHVL×D8 VH-A240VL# (SEQ ID NO. 136),

and SEQ ID NO.77 VHVL×CEAI VH-A240VL (SEQ ID NO. 20).

(ii) VL-VH orientation of anti-CEA:

SEQ ID NO.77 VHVL×A240VL-A5 VH (SEQ ID NO. 28),

SEQ ID NO.77 VHVL×A240VL-B9VH (SEQ ID NO. 38),

SEQ ID NO.77 VHVL×A240VL-D8VH (SEQ ID NO. 46), and

SEQ ID NO.77 VHVL×A240VL-CEAI VH (SEQ ID NO. 22).

CEAI VH (SEQ ID NO. 56) denotes a VH region derived from murine mAbA5B7, whereas CEAI VHVL or CEAI VLVH refer to a VH-VL domain and a VL-VHdomain, respectively, derived from mAb A5B7. A240 corresponds to a humanVL region (see SEQ ID NO. 64 and Example 7). Accordingly, e.g. CEAIVH-A240 VL×SEQ ID NO.77 VHVL (SEQ ID NO.16) corresponds to a bispecificconstruct with a half-human CEA-binding domain having a murine VH regionfrom mAb7 and a human VL region A240. For the cloning of the humanantibody library, the nucleotide sequences encoding the originalN-termini of the A240 VL region had to be converted to restrictionsites. In A5 VH-A240 VL#×SEQ ID NO.77 VHVL (SEQ ID NO. 126), SEQ IDNO.77 VHVL×A5 VH-A240VL# (SEQ ID NO. 128), B9 VH-A240 VL#×SEQ ID NO.77VHVL (SEQ ID NO. 130), SEQ ID NO.77 VHVL×B9 VH-A240VL# (SEQ ID NO. 132),D8 VH-A240 VL#×SEQ ID NO.77 VHVL (SEQ ID NO. 134), and SEQ ID NO.77VHVL×D8 VH-A240VL# (SEQ ID NO. 136), the original N-termini have beenreintroduced.

The bispecific single chain antibody construct SEQ ID NO.77 VHVL×E12VH-A240VL (SEQ ID NO. 143) differs from the SEQ ID NO.77 VHVL×B9VH-A240VL (SEQ ID NO. 36) construct in only one amino acid residue: TheCDR-H2 sequence in the E12 VH reads “FILNKANGGTTEYAASVKG” (SEQ ID NO.145), whereas in the B9 VH it reads “FIRNKANGGTTEYAASVKG” (SEQ ID NO.67). The human E12 VH region has been isolated as set forth in Example7.

2. Expression, Purification and Flow Cytometry Analysis

Expression, purification and flow cytometry analysis of these humanCEA×CD3 bispecific single chain antibodies has been carried out bymethods described in Example 2, supra.

3. Binding Activity

As exemplified in FIG. 14, human bispecific single chain antibodyconstructs A5 VH-A240 VL×SEQ ID NO.77 VHVL (SEQ ID NO. 24), B9 VH-A240VL×SEQ ID NO.77 VHVL (SEQ ID NO. 32), D8 VH-A240 VL×SEQ ID NO.77 VHVL(SEQ ID NO. 40) and CEAI VH-A240 VL×SEQ ID NO.77 VHVL (SEQ ID NO. 16)bind to CEA-positive human Kato III cells and to CD3-positive HPB-ALLcells.

4. Cytotoxic Activity in the Absence of Soluble CEA Antigen

Cytotoxic activity against CHO-CEA+ target cells has been demonstratedfor the following domain arrangements of the human bispecific singlechain antibodies:

For constructs with the anti-CEA part located N-terminally and a VH-VLorientation of the anti-CEA part, FIG. 15 shows cytotoxic activity forA5 VH-A240 VL×SEQ ID NO.77 VHVL (SEQ ID NO. 24), B9 VH-A240 VL×SEQ IDNO.77 VHVL (SEQ ID NO. 32), D8 VH-A240 VL×SEQ ID NO.77 VHVL (SEQ ID NO.40) and CEAI VH-A240 VL×SEQ ID NO.77 VHVL (SEQ ID NO. 16). FIG. 16demonstrates cytotoxic activity for the VL-VH orientation of theanti-CEA domain (located N-terminally) for A240 VL-A5 VH×SEQ ID NO.77VHVL (SEQ ID NO. 26), A240 VL-B9 VH×SEQ ID NO.77 VHVL (SEQ ID NO. 34),A240 VL-D8 VH×SEQ ID NO.77 VHVL (SEQ ID NO. 42), and A240 VL-CEAI VH×SEQID NO.77 VHVL (SEQ ID NO. 18).

For constructs with the anti-CEA part located C-terminally and a VH-VLorientation of the anti-CEA part, FIG. 17 exhibits cytotoxicity againstCEA+ target cells for SEQ ID NO.77 VHVL×A5 VH-A240VL (SEQ ID NO. 30),SEQ ID NO.77 VHVL×B9 VH-A240VL (SEQ ID NO. 36), SEQ ID NO.77 VHVL×D8VH-A240VL (SEQ ID NO. 44), and SEQ ID NO.77 VHVL×CEAI VH-A240VL (SEQ IDNO. 20). SEQ ID NO.77 VHVL×CEAI LH was used as a positive control. FIG.18 shows cytotoxic activity of constructs with VL-VH orientation of theanti-CEA domain (located C-terminally) for SEQ ID NO.77 VHVL×A240VL-A5VH (SEQ ID NO. 28), SEQ ID NO.77 VHVL×A240VL-B9VH (SEQ ID NO. 38), SEQID NO.77 VHVL×A240VL-D8VH (SEQ ID NO. 46), and SEQ ID NO.77VHVL×A240VL-CEAIVH (SEQ ID NO. 22). SEQ ID NO.77 VHVL×CEAI LH (SEQ IDNO. 2) was used as a positive control.

FIG. 21 shows cytotoxic activity of bispecific single chain antibodyconstructs comprising the human B9 VH region and the human A240 VLregion in different arrangements: A240 VL-B9 VH×SEQ ID NO.77 VHVL (SEQID NO. 34), SEQ ID NO.77 VHVL×A240 VL-B9 VH (SEQ ID NO. 38), SEQ IDNO.77 VHVL×B9 VH-A240 VL (SEQ ID NO. 36), and B9 VH-A240 VL×SEQ ID NO.77VHVL (SEQ ID NO. 32) revealed cytotoxic activity against humanCEA-transfected CHO cells.

5. Resistance of Human CEA×CD3 Bispecific Single Chain Antibodies toSoluble CEA Antigen

Competition assays in the presence of soluble human CEA antigen for thehuman bispecific single chain antibodies have been performed as set outin Example 5, supra. FIGS. 19 and 20 demonstrate the resistance tosoluble CEA antigen of cytotoxic activity also for human constructs, asexemplified for constructs A5 VH-A240 VL×SEQ ID NO.77 VHVL (SEQ ID NO.24), B9 VH-A240 VL×SEQ ID NO.77 VHVL (SEQ ID NO. 32), D8 VH-A240 VL×SEQID NO.77 VHVL (SEQ ID NO. 40) and CEAI VH-A240 VL×SEQ ID NO.77 VHVL (SEQID NO. 16); for resistance to soluble CEA of murine-derived bispecificsingle chain antibodies see Example 5. Moreover, FIG. 22 shows thatCEA-reactive bispecific single chain construct A240 VL-B9 VH×SEQ IDNO.77 VHVL (SEQ ID NO. 34) redirected T cells to lyse CHO-CEA+ cells inthe presence of increasing amounts of soluble CEA antigen. Thisexperiment demonstrates that also A240 VL-B9 VH×SEQ ID NO.77VHVL-mediated cytotoxic activity is resistant to soluble CEA. Finally,as can be derived from FIG. 27, bispecific single chain construct SEQ IDNO.77 VHVL×E12 VH-A240 VL (SEQ ID NO. 143)-mediated cytotoxic activityis resistant to soluble CEA.

Thus, the present invention provides for pharmaceutical compositionswith cytotoxic anti-tumor activity in the presence of even high levelsof soluble CEA antigen. As set forth above, high serum CEAconcentrations in patients with epithelial tumors reduce the success ofanti-CEA antibody-based therapeutics. Therefore, the pharmaceuticalcompositions of the invention are particularly suitable for thetreatment of patients with progressive epithelial tumors (e.g.secondary, metastatic tumors after surgical removal of the primarytumor(s)), malignant epithelial tumors, high (epithelial) tumor load andlate stage epithelial tumors characterized by high soluble CEA antigenconcentrations in the serum/plasma of said patients. Further,pharmaceutical compositions of the invention are also expected to beadministered at low dosages. In addition, said pharmaceuticalcompositions are unlikely to be immunogenic when administered to thepatients due to the human origin of the anti-CEA part and thede-immunised anti-CD3 part of the bispecific single chain antibodies inthe pharmaceutical compositions of the invention. Moreover, thepharmaceutical compositions of the invention are expected to provide forhigh tumor penetration due to the low molecular weight and small size ofthe bispecific single chain constructs. Additionally, for the tumortreatment low amounts of bispecific single chain antibodies will be usedbecause of the high cytotoxic activity of said molecules. Because lowamounts are expected to be administered to the patients, the adverseeffects for the patients are also expected to be reduced. Finally,bispecific single chain antibodies without resistance to soluble CEAwould be highly sensitive to even low concentrations of soluble CEAantigen in the plasma of tumor patients since they would be administeredin low concentrations, as set forth above. This problem is alsocircumvented by the pharmaceutical compositions of the invention.

EXAMPLE 9: PROTEIN HOMOGENEITY ASSESSMENT OF PURIFIED MONOMER OF A240VL-B9 VH×SEQ ID NO.77 VHVL (SEQ ID NO. 34) BY HIGH RESOLUTION CATIONEXCHANGE CHROMATOGRAPHY

To further characterize the homogeneity of the purified A240 VL-B9VH×SEQ ID NO.77 VHVL (SEQ ID NO. 34) construct, the isolated monomerfraction was subjected to a High Resolution Cation ExchangeChromatography. The chromatography was performed on a MiniS column (MiniS PE 4.6/50 CatX 0.8 ml; GE Healthcare 17-5005-01), equilibrated with 20mM MES buffer pH 5.5. The sample was diluted 1:3 with the same bufferbefore loading to the column. Bound protein was eluted with a gradientof equilibration buffer containing 1M NaCl: 0-30% in 60 column volumes.Remaining protein was eluted in 3 column volumes of 1M NaCl. Theresulting chromatogram is shown in FIG. 23 and exhibits a homogenousprotein fraction with a single main peak.

EXAMPLE 10: PLASMA STABILITY OF A240 VL-B9 VH×SEQ ID NO.77 VHVL (SEQ IDNO. 34)

The plasma stability of the A240 VL-B9 VH×SEQ ID NO.77 VHVL (SEQ ID NO.34) construct was tested under different incubation conditions followedby a standard 51-chromium release based cytotoxicity assay as describedin Example 4.

A human plasma pool with the blood of five healthy donors was generatedby collecting blood in EDTA-coated syringes. The cellular componentswere removed by centrifugation and the upper plasma phase was collectedand subsequently pooled. The A240 VL-B9 VH×SEQ ID NO.77 VHVL (SEQ ID NO.34) construct was either incubated at 37° C. or 4° C. in the presence orabsence of plasma. As controls, the construct was diluted immediatelyprior to the cytotoxicity assay in plasma or RPMI 1640 medium,respectively. CHO-CEA+ served as target cells; stimulated CD8+ T cellswere used as effector cells. The effector:target (E:T) ratio was 10:1.The assay duration was 18 hours.

As shown in FIG. 24, the CEA-reactive bispecific single chain constructA240 VL-B9 VH×SEQ ID NO.77 VHVL (SEQ ID NO. 34) proved to be verystable; no loss of cytotoxic activity could be detected after incubationin human plasma for 24 hours at 37° C.

EXAMPLE 11: PROTEIN HOMOGENEITY ASSESSMENT OF PURIFIED MONOMER OF SEQ IDNO.77 VHVL×E12 VH-A240 VL (SEQ ID NO. 143) BY HIGH RESOLUTION CATIONEXCHANGE CHROMATOGRAPHY

The experiment was conducted in analogy with Example 9, except that SEQID NO.77 VHVL×E12 VH-A240 VL (SEQ ID NO. 143) was analyzed. Theresulting chromatogram is shown in FIG. 25 and exhibits a homogenousprotein fraction with a single main peak.

EXAMPLE 12: PLASMA STABILITY OF SEQ ID NO.77 VHVL×E12 VH-A240 VL (SEQ IDNO. 143)

The experiment was conducted in analogy with Example 10, except that SEQID NO.77 VHVL×E12 VH-A240 VL (SEQ ID NO. 143) was analyzed. As displayedin FIG. 26, the CEA-reactive bispecific single chain construct SEQ IDNO.77 VHVL×E12 VH-A240 VL (SEQ ID NO. 143) proved to be very stable; noloss of cytotoxic activity could be detected after incubation in humanplasma for 24 hours at 37° C.

The invention claimed is:
 1. A bispecific single chain antibodycomprising (a) a first binding domain specifically binding to human CD3,and (b) a second binding domain specifically binding to human CEA,wherein said second binding domain comprises a variable light chain(V_(L)) region and a variable heavy chain (V_(H)) region; wherein saidV_(L) region comprises a CDR-L1 having the amino acid sequence“TLRRGINVGAYSIY” (SEQ ID NO: 73), a CDR-L2 having the amino acidsequence “YKSDSDKQQGS” (SEQ ID NO: 72), and a CDR-L3 having the aminoacid sequence “MIWHSGASAV” (SEC) ID NO: 71); and wherein said V_(H)region comprises a CDR-H1 having the amino acid sequence “SYWMH” (SEQ IDNO: 68), a CDR-H2 having the amino acid sequence “FILNKANGGTTEYAASVKG”(SEC) ID NO: 145), and a CDR-H3 having the amino acid sequence“DX₁X₂X₃X₄FYFDY” (SEQ ID NO: 65), wherein “X₁” is “R” (Arginine), “F”(Phenylalanine), “M” (Methionine), “E” (Glutamic acid), or “T”(Threonine), “X₂” is “G” (Glycine), “Y” (Tyrosine), “A” (Alanine), “D”(Aspartic acid), or “S” (Serine), “X₃” is “L” (Leucine), “F”(Phenylalanine), “M” (Methionine), “E” (Glutamic acid), or “T”(Threonine), and “X₄” is “R” (Arginine), “Y” (Tyrosine), “A” (Alanine),“D” (Aspartic acid), or “S” (Serine), and the amino acid residue “D”corresponds to Kabat position 95 of CDR-H3 of murine monoclonal antibodyA5B7, and the amino acid residues “FYFDY” correspond to Kabat positions100, 100a, 100b, 101, and 102, respectively, of CDR-H3 of murinemonoclonal antibody A5B7.
 2. The bispecific single chain antibody ofclaim 1, wherein said CDR-H3 comprises at least the amino acid sequence“DRGLRFYFDY” (SEC) ID NO: 66).
 3. The bispecific single chain antibodyof claim 1, wherein said first binding domain is located C-terminally tosaid second binding domain.
 4. The bispecific single chain antibody ofclaim 1, wherein said binding domains are arranged in the orderVH_(CEA)-VL-_(CEA)-VH_(CD3)-VL_(CD3) orVL_(CEA)-VH_(CEA)-VH_(CD3)-VL_(CD3).
 5. The bispecific single chainantibody of claim 1, wherein the amino acid sequence of the VH region ofthe second binding domain is set forth in SEQ ID NO:
 146. 6. Thebispecific single chain antibody of claim 1, wherein the amino acidsequence of the VL region of the second binding domain is set forth inSEQ ID NO:
 64. 7. The bispecific single chain antibody of claim 1,wherein the second binding domain comprises the VH region comprising theamino acid sequence set forth in SEQ ID NO: 146 and the VL regioncomprising the amino acid sequence set forth in SEQ ID NO:
 64. 8. Thebispecific single chain antibody of claim 1 comprising an amino acidsequence selected from the group consisting of: (a) the amino acidsequence set forth in SEQ ID NO: 143; (b) the amino acid sequenceencoded by the nucleic acid sequence as set forth in SEQ ID NO: 142; (c)an amino acid sequence encoded by a nucleic acid sequence which isdegenerate as a result of the genetic code to a nucleotide sequence of(b); and (d) an amino acid sequence at least 85% identical, at least 90%identical, or at least 95% identical to the amino acid sequence of (a)or (b).
 9. The bispecific single chain antibody of claim 1, wherein atleast one of said first or second binding domains is chimeric,humanized, CDR-grafted, deimmunized or human.
 10. A pharmaceuticalcomposition comprising (a) the bispecific single chain antibody of claim1; (b) a nucleic acid molecule encoding the bispecific single chainantibody of claim; (c) a vector comprising the nucleic acid molecule of(b); or (d) a host cell transformed or transfected with the nucleic acidmolecule of (b) or the vector of (c), and a carrier, stabilizer, orexcipient.
 11. The pharmaceutical composition of claim 10,further-comprising a proteinaceous compound capable of providing anactivation signal for immune effector cells.
 12. A process for producingthe bispecific single chain antibody of claim 1, said process comprisingculturing a host cell transformed or transfected with a nucleic acidmolecule encoding the bispecific single chain antibody or with a vectorcomprising the nucleic acid molecule under conditions allowing theexpression of the bispecific single chain antibody, and, optionally,recovering the bispecific single chain antibody from the culture.
 13. Amethod for treating an epithelial tumor expressing CEA in a subject,said method comprising administering an effective amount of thebispecific single chain antibody of claim
 1. 14. The method of claim 13,wherein said epithelial tumor is a gastrointestinal adenocarcinoma, abreast adenocarcinoma or a lung adenocarcinoma.
 15. The method of claim14, wherein said gastrointestinal adenocarcinoma is a colorectaladenocarcinoma, a pancreatic adenocarcinoma, an oesophagealadenocarcinoma, or a gastric adenocarcinoma.
 16. The method claim 13,wherein the subject suffers from a progressive tumor, a late stagetumor, a high tumor load or burden, a metastatic tumor, or a CEA serumconcentration higher than 100 ng/ml.
 17. The method of claim 13, whereinsaid bispecific single chain antibody is administered in combinationwith an additional drug.
 18. The method of claim 17, wherein saidadditional drug is a proteinaceous compound.
 19. The method of claim 18,wherein the proteinaceous compound is capable of providing an activationsignal for immune effector cells.
 20. The method of claim 17, whereinsaid additional drug is administered simultaneously with the bispecificsingle chain antibody.
 21. The method of claim 13, wherein said subjectis a human.
 22. A kit comprising the bispecific single chain antibody ofclaim 1, a nucleic acid molecule encoding such bispecific single chainantibody, a vector comprising such nucleic acid molecule, or a host celltransformed or transfected with such nucleic acid or vector.
 23. Themethod of claim 16, wherein said CEA serum concentration is determinedby ELISA.
 24. The method of claim 17, wherein said additional drug is anon-proteinaceous compound.
 25. The method of claim 17, wherein saidadditional drug is administered non-simultaneously with the bispecificsingle chain antibody.